Journal of Thrombosis and Haemostasis

Anti-prothrombin antibodies are commonly found in patients with Antiphospholipid Syndrome (APS), yet their role in clinical manifestations remains unclear. We recently identified two classes of anti-prothrombin antibodies based on their ability to recognize closed and open forms of prothrombin. Type-I antibodies bind to the open form, while Type-II antibodies bind to both forms. POmAb is a prototypical Type-I antibody that specifically targets kringle-1 of prothrombin, maintaining it in an open state. In this study, we assess the effects of POmAb in mice using the cremaster arteriole laser-induced injury model. POmAb bound mouse prothrombin and decreased thrombin generation in mouse plasma. When administered intravenously shortly before the injury, POmAb quickly accumulated on the damaged vessel wall. This accumulation significantly reduced fibrin generation with a modest effect on platelet accumulation and without causing excessive bleeding. Results obtained with POmAb offer insights into the potential roles of the anti-prothrombin antibodies in APS. They also provide proof of concept for a new class of anticoagulants that, by specifically targeting open prothrombin, could mitigate thrombosis with reduced bleeding risk.

BackgroundPost-acute sequelae of COVID-19 (PASC), also referred as Long-COVID, sometimes follows COVID-19, a disease caused by SARS-CoV-2. While SARS-CoV-2 is well-known to promote a prothrombotic state, less is known about the thrombosis risk in PASC. AimOur objective was to evaluate the platelet function and thrombotic potential in patients following recovery from SARS-CoV-2 with clear symptoms of PASC. MethodsPASC patients and matched healthy controls were enrolled in the study on average 15 months after documented SARS-CoV-2 infection. Platelet activation was evaluated by Light Transmission Aggregometry (LTA) and flow cytometry in response to platelet surface receptor agonists. Thrombosis in platelet-deplete plasma was evaluated by Factor Xa activity. A microfluidics system assessed thrombosis in whole blood under shear stress conditions. ResultsA mild increase in platelet aggregation in PASC patients through the thromboxane receptor was observed and platelet activation through the glycoprotein VI (GPVI) receptor was decreased in PASC patients compared to age- and sex-matched healthy controls. Thrombosis under shear conditions as well as Factor Xa activity were reduced in PASC patients. Plasma from PASC patients was an extremely potent activator of washed, healthy platelets - a phenomenon not observed when stimulating healthy platelets after incubation with plasma from healthy individuals. ConclusionsPASC patients show dysregulated responses in platelets and coagulation in plasma, likely caused by a circulating molecule that promotes thrombosis. A hitherto undescribed protective response appears to exists in PASC patients to counterbalance ongoing thrombosis that is common to SARS-CoV-2 infection.

BackgroundPatients with coronavirus disease-19 (COVID-19) are at increased risk of thrombosis, which is associated with altered platelet function and coagulopathy, contributing to excess mortality. ObjectivesWe aimed to characterise the mechanism of altered platelet function in COVID-19 patients. MethodsThe platelet proteome, platelet functional responses and platelet-neutrophil aggregates were compared between patients hospitalised with COVID-19 and healthy control subjects using Tandem Mass Tag (TMT) proteomic analysis, Western blotting and flow cytometry. ResultsCOVID-19 patients showed a different profile of platelet protein expression (858 altered out of 5773 quantified). Levels of COVID-19 plasma markers were enhanced in COVID-19 platelets. Gene ontology (GO) pathway analysis demonstrated that levels of granule secretory proteins were raised, whereas some platelet activation proteins, such as the thrombopoietin receptor and PKC, were lowered. Basally, COVID-19 platelets showed enhanced phosphatidylserine (PS) exposure, with unaltered integrin IIb{beta}3 activation and P-selectin expression. Agonist-stimulated integrin IIb{beta}3 activation and PS exposure, but not P-selectin expression, were significantly decreased in COVID-19 patients. COVID-19 patients had high levels of platelet-neutrophil aggregates, even under basal conditions, compared to controls. This interaction was disrupted by blocking P-selectin, demonstrating that platelet P-selectin is critical for the interaction. ConclusionsOverall, our data suggests the presence of two platelet populations in patients with COVID-19: one with circulating platelets with an altered proteome and reduced functional responses and another with P-selectin expressing neutrophil-associated platelets. Platelet driven thromboinflammation may therefore be one of the key factors enhancing the risk of thrombosis in COVID-19 patients. Essentials- COVID-19 patient platelet function and platelet proteins were compared with healthy controls - Proteomic analysis of platelets indicated that COVID-19 decreased platelet activation proteins - Agonist induced PS exposure and integrin IIb{beta}3 activation were impaired in COVID-19 - COVID-19 led to maximal levels of P-selectin dependent platelet-neutrophil aggregates

BackgroundPlatelets are crucial mediators at the crossroads of hemostasis, immunity, and inflammation, adapting their responses to diverse stimuli. Despite their recognized role, the precise pathways and markers associated with platelet activation remain poorly understood. This study aimed to unravel these mechanisms by evaluating platelet responses to various agonists, employing biomathematical models to map activation patterns and identify key biomarkers. MethodsUsing samples from ten healthy donors, platelets were exposed to seven stimulation conditions: unstimulated, PAR-1 agonist TRAP, PAR-4 agonist AYPGKF, ADP, collagen, sCD40L, and fibrinogen. A comprehensive analysis of 47 biological markers--covering membrane activation, soluble mediators, and signaling pathways--was conducted. Statistical and machine learning models, including hierarchical clustering and random forests, were applied to classify and interpret platelet activation signatures. ResultsDistinct activation profiles were observed for each agonist. A streamlined panel of six markers--AKT, CD40 ligand, CD62P (mean fluorescence intensity and percentage), PKC, RANTES, and TSLP--achieved 86.8% accuracy in identifying the activating stimulus. The study highlighted significant variations, influenced by both the stimulus and donor-specific factors. Machine learning approaches further refined classification, achieving a multiclass accuracy of 87.9%. Hierarchical clustering demonstrated clear distinctions, particularly between PAR-1/PAR-4 responses and other agonists. ConclusionThis innovative research redefines platelets as dynamic "biological sensors" capable of decoding complex danger signals. By integrating biomathematical modeling and artificial intelligence, it identifies a precise biomarker panel with transformative potential for diagnostics and therapies in inflammation and immune disorders. This work positions platelets not just as key players in hemostasis but as programmable agents for precision medicine, heralding a new era in adaptive, AI-driven healthcare solutions. Author SummaryPlatelets are often seen as simple players in blood clotting, but they do much more. They sit at the crossroads of hemostasis (stopping bleeding), innate immunity (our bodys first defense), and inflammation. They even influence adaptive immunity and play key roles in maintaining healthy blood vessels and contributing to disease. What makes platelets fascinating is their ability to respond quickly to their environment. They carry various receptors and release substances like growth factors, immune signals, clotting factors, RNA, and tiny vesicles. This helps them react to threats and communicate with other cells. But the big question is: can platelets tailor their response based on specific stimuli? In my research, I set out to answer this. Using mathematical models and analytical techniques, I studied how platelets react to different triggers, especially those linked to immune and clotting responses. My goal was to identify specific molecular "signatures" that define how platelets respond. I found that platelets can distinguish between danger signals and adjust their secretory responses. Normally, this helps manage threats efficiently. However, when this response exceeds whats needed, it can contribute to diseases like cardiovascular disorders, severe infections, autoimmune conditions, and cancer. Understanding these pathways opens new doors for treatment. Since platelet activity can be influenced by drugs, we could shift their role from harmful to beneficial in many diseases. This could revolutionize how we approach conditions driven by inflammation and immune dysregulation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/636809v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@9d9b5forg.highwire.dtl.DTLVardef@1438071org.highwire.dtl.DTLVardef@a59b29org.highwire.dtl.DTLVardef@6dd66b_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundComplement FH (FH) is a key regulator of complement activity whereas thrombin (FIIa) is central to hemostasis with both pro- and anticoagulant functions. Both have separately been shown to have auxiliary activities across the two systems. The purpose of this study was to determine the effect of FH on pro- and anti-coagulant functions and investigate the interaction between FH and thrombin. MethodsTail bleeding time and hemolysis were measured in FH-deficient mice (CFH-/-). Activated partial thromboplastin time (aPTT) was determined in FH-depleted human plasma. FH effect on fibrin clot generation was investigated in turbidity assays and on activated protein C (APC) generation. Binding affinity of thrombin with FH was determined using surface plasmon resonance (SPR). ResultsTail bleeding time in CFH-/- mice was significantly prolonged compared to wild type mice. The aPTT in FH-depleted human plasma was elevated compared to normal plasma and restored by adding back FH to depleted plasma. Accordingly, FH enhanced thrombin-mediated fibrin clot generation by shortening lag time, increasing rate of clot formation and maximum turbidity, and affected clot structure. Despite this, FH also increased the rate of thrombin-mediated protein C (PC) activation, both in the presence and absence of soluble recombinant thrombomodulin (TM). Nanomolar affinity binding of FH with thrombin, but not prothrombin, was confirmed. ConclusionComplement FH binds thrombin with strong affinity and acts as a novel cofactor that enhances both pro- and anticoagulant actions of thrombin. These data highlight an important role for FH in hemostasis. Key pointsO_LIAbsence of FH prolongs tail bleeding time in CFH-/- mice and activated partial thromboplastin time (aPTT) is elevated in human FH-depleted plasma. C_LIO_LIFH acts a cofactor for thrombin by enhancing fibrin generation, altering fibrin clot structure and enhancing TM-thrombin mediated protein C activation C_LI

PurposeA central role for the pro-coagulant membrane comprising aminophospholipids (aPL) and enzymatically oxidized phospholipids (eoxPL) in promoting hemostasis via interaction with coagulation factor Gla domains is well established. However, little is known about their interactions with the fibrinolytic pathway, their ability to alter clot structure or to support the activated protein C (APC) pathway. Previous studies used membrane liposome compositions that differ from those expected physiologically and/or generated inconsistent findings. To address this, pro-coagulant membranes comprising physiological proportions of aPL and eoxPL will be tested for their ability to support fibrinolysis using standard assays. MethodsThe impact of phospholipids on clot structure and clot lysis was tested using absorbance-based assays. To investigate the impact of PS or eoxPL on fibrinolysis, plasmin was monitored chromogenically, and clot dissolution measured in a purified lysis system activated by tissue plasminogen activator or urokinase. To determine the impact of eoxPL on APC/protein S, FVa was incubated with APC (+/-protein S) in a purified prothrombinase assay. ResultsAt the concentrations of lipids tested in our study, PS did not significantly impact clot structure or fibrinolysis. Similarly, eoxPL did not impact either fibrinolysis or activity of APC/Protein S. ConclusionUsing liposome compositions that approximate activated blood cells, we found that the pro-coagulant membrane is unlikely to influence either clot structure or fibrinolytic activity directly, beyond its well characterized role in supporting Gla dependent coagulation factors and the actions of platelet associated proteins/receptors.

Bleeding patterns in people with hemophilia A cannot be predicted solely by factor VIII (FVIII) levels. Some of the variance in bleeding may be attributed to differences in plasma protein composition, and specifically other coagulation factors where the normal ranges span 50-150% of the population mean. We recently used a mathematical model of thrombus formation that identified factor V (FV) levels as a strong modifier of thrombin generation in FVIII deficiencies. Counterintuitively, the model predicted low normal FV levels enhanced thrombin generation. Here, we tested this prediction and investigated its mechanism. Thrombin generation in plasma from people with FVIII deficiencies (<5%) were negatively correlated with FV levels. A substrate competition mechanism wherein FV and FVIII compete for activation by FXa during the initiation of coagulation was tested in three models: In a purified system containing only FV, FVIII, and FXa, reducing FV enhanced FVIII activation. In synthetic plasma containing the essential proteins of the extrinsic coagulation pathway, low normal FV levels resulted in enhanced thrombin generation both in the presence or absence of TFPI. In mixture studies using FVIII-deficient human plasma immunodepleted of FV, thrombin generation was enhanced at lower levels of FV. In all models the trend was nonlinear as the effect size was significant at low, but not high, FV levels. Our data show that low normal plasma levels of FV enhance thrombin generation in hemophilia A by reducing FXa substrate competition for FVIII activation and implicate FV levels as a strong modifier of bleeding in hemophilia A. Key PointsO_LILow normal levels of FV enhance thrombin generation in hemophilia A by reducing substate competition for FVIII activation. C_LIO_LIPlasma FV levels are a strong modifier of bleeding in hemophilia A. C_LI

BackgroundTraumatic injury is a leading cause of death for those under the age of 45, with 40% occurring due to hemorrhage. Severe tissue injury and hypoperfusion lead to marked changes in coagulation, thereby preventing formation of a stable blood clot and increasing hemorrhage associated mortality. ObjectivesWe aimed to quantify changes in clot formation and mechanics occurring after traumatic injury and the relationship to coagulation kinetics, and fibrinolysis. MethodsPlasma was isolated from injured patients upon arrival to the emergency department. Coagulation kinetics and mechanics of healthy donors and patient plasma were compared with rheological, turbidimetric and thrombin generation assays. ELISAs were performed to determine tissue plasminogen activator (tPA) and D-dimer concentration, as fibrinolytic markers. ResultsSixty-three patients were included in the study. The median injury severity score (ISS) was 17, median age was 37.5 years old, and mortality rate was 30%. Rheological, turbidimetric and thrombin generation assays indicated that trauma patients on average, and especially deceased patients, exhibited reduced clot stiffness, increased fibrinolysis and reduced thrombin generation compared to healthy donors. Fibrinogen concentration, clot stiffness, D-dimer and tPA all demonstrated significant direct correlation to increasing ISS. Machine learning algorithms identified and highlighted the importance of clinical factors on determining patient outcomes. ConclusionsViscoelastic and biochemical assays indicate significant contributors and predictors of mortality for improved patient treatment and therapeutic target detection. ESSENTIALSO_LITraumatic injury may lead to alterations in a patients ability to form stable blood clots C_LIO_LIA study was performed to assess how trauma severity affects coagulation kinetics C_LIO_LIKey alterations were observed in trauma patients, who exhibit weaker and slower forming clots C_LIO_LIPaired with machine learning methods, the results indicate key aspects contributing to mortality C_LI

Platelet adhesion and procoagulant activity are critical for primary and secondary hemostasis, respectively. The small GTPase RAP1 is a central regulator of platelet aggregation as it controls IIb{beta}3 integrin activation through direct interaction with the integrin adapter protein, TALIN-1 (Tln-1). In addition to their aggregation defect, activated platelets lacking RAP1 (Rap1mKO) exhibited a marked impairment in surface exposure of phosphatidylserine (PtdSer), a negatively charged phospholipid with procoagulant activity. However, the mechanisms by which RAP1 regulates PtdSer exposure are unclear. Here we investigated the hypothesis that RAP1 regulates platelet PtdSer exposure through cross-talk with small GTPases of the Rho family. Consistent with their defect in PtdSer exposure, Rap1mKO platelets showed reduced procoagulant activity in vitro and in vivo when compared to controls. Stimulated Rap1mKO platelets exhibited elevated RHOA-GTP levels, and inhibition of the RHOA effector, Rho associated coiled-coil kinase (ROCK), partially restored PtdSer exposure in these cells. A milder defect in PtdSer exposure was observed for platelets from Tln-1mR35/118E mice, i.e. mice with impaired RAP1-Tln-1 interaction but otherwise intact RAP1 signaling. ROCK inhibition fully restored PtdSer exposure in Tln-1mR35/118E platelets. Opening of the mitochondrial permeability transition pore, a cellular response critical to PtdSer exposure, was impaired in Rap1mKO platelets and restored by pretreatment of cells with the ROCK inhibitor. Our study provides first evidence that platelet RAP1 signaling affects hemostatic plug formation independent of its key role in platelet adhesion. Additionally, our studies strongly suggest that RAP1 regulates PtdSer exposure and procoagulant activity in a RHOA/integrin-dependent and -independent manner.

BackgroundThe contributions of platelet activation to thrombus formation during hemostatic bleeding cessation likely involve multiple activation states. However, the spatial and temporal distribution of platelets in these states has not been defined in a clot. ObjectivesTo use single-platelet mapping of activation states within jugular vein puncture thrombi to determine how the spatial distribution of platelet state evolves during hemostasis. MethodsMontaged, wide-area electron micrographs (EM) were taken at various time points, post-puncture, and annotated for platelet activation state. These classifications were mapped onto the images to identify regions of platelet activation and calculate neighbor associations. The importance of -granule secretion was tested using VAMP8-/- mice. Resultsmapping of platelet activation states at 1 min post-puncture showed extensive spatial intermixing of most platelet activation classes. No high-activation-state, platelet-rich core was observed in 5-min post-puncture thrombi, rather such platelets tended to be localized on the interior surfaces of thrombus vaults open to the circulation. Only at a later stage, 20 min post-puncture, was distinct clustering of high activation, degranulated, cytosol-rich platelets observed. These clusters localized to the central portion of the intravascular platelet-rich crown, and they were now inaccessible to the circulation. Counterintuitively, deletion of the primary platelet v-SNARE, VAMP8, increased the frequency and spatial clustering of highly activated, degranulated platelets in association with intra-thrombus vault surfaces at 5 min post puncture. ConclusionsWe conclude recent multi-activation state models can provide a realistic thrombus formation framework if linked together to encompass the dynamics of puncture wound formation.

Background: Uncontrolled bleeding complicates trauma, surgery and many medical conditions. While currently available procoagulant therapies (e.g., plasma-derived factors, recombinant proteins, antifibrinolytics) have crucial limitations. Methods: N389 (CQQTVTLLPAADLDDFSC) was synthesized by Fmoc solid-phase chemistry, characterized by HPLC and LC-MS, then tested in normal human pooled plasma in microplate mechanical clot-formation assays using incubated and immediate addition formats. Kinetic parameters (plasma recalcification, PRT; maximum absorbance, Amax) were obtained from absorbance curves fit to four-parameter logistic models. Mixing studies with modified (i.e., aged, adsorbed) plasma probed factor dependence. Results: In plasma coagulation assays activated with 25 mM CaCl2, baseline clotting showed a PRT of 23.74 +/- 0.27 min and Amax of 0.1813 +/- 0.0043 (n = 3), whereas N389 significantly reduced PRT to 8.442 +/- 6.0395 min without incubation (p = 0.0012), further decreased PRT after incubation (p < 0.0001), increased Amax to 0.2523, and retained comparable activity across normal, adsorbed, and aged plasma, in contrast to S1255 which showed a faster but incubation-labile effect with PRT 2.353 +/- 1.3685 min (p = 0.0007) and marked attenuation in factor-depleted and aged plasma. Mixing studies showed N389 activity persisted across normal, aged and adsorbed plasma, consistent with a mechanism that does not require intact plasma coagulation factor profiles (specifically factors II, V, VIII, VII, IX, X). Discussion: Collectively with prior evidence on anionic surfaces, Ca2+-binding Gla domains, and peptide-modulated fibrin polymerization, these results support a model in which N389 functions as a stable, charge-based scaffold that coordinates divalent cations and/or directly nucleates fibrin(ogen), while highlighting limitations of bulk clotting assays and the need for targeted thrombin generation, binding, aggregation, and contact-activation studies. Conclusions: The aspartate-rich peptide N389 is a sustained, factor-independent procoagulant at least in vitro. N389 thus merits further mechanistic and translational evaluation as a synthetic hemostatic agent.

Snakebite globally claims more than 100,000 lives per year and results in morbidity for 400,000 survivors. Current treatment uses antibody-based antivenoms which are constrained by their efficacy, safety and cost. In this study we evaluated the efficacy of previously described repurposed drugs against viperid snakes of the medically important Bothrops genus. Despite variable toxin representation and bioactivity across this central and south American genus, we found that the lead inhibitors targeting metalloproteinases (marimastat and DMPS) and phospholipases (varespladib), demonstrated pan-species neutralisation in enzymatic assays, whilst nafamostat (serine protease inhibitor) had variable activity. The metalloproteinase inhibitors protected against the procoagulant and haemorrhagic effects of several venoms in phenotypic assays. Collectively these findings demonstrate that repurposed drugs may be of great value as early interventions for the treatment of bothropic envenoming in the Neotropics and thus provides a strong rationale for their progression into future preclinical and clinical evaluation for snakebite indication.

In vulnerable atherosclerotic plaques intraplaque hemorrhages (IPH) result in hemolysis of red blood cells and release of hemoglobin and free hemin. Hemin activates platelets and leads to thrombosis. Agonism of the inhibitory platelet receptor ACKR3 inhibits hemin-dependent platelet activation and thrombus formation. To characterize the effect of hemin and ACKR3 agonism on isolated human platelets, multi-color flow cytometry and classical experimental setup such as light transmission aggregometry and a flow chamber assay have been used. Hemin induces platelet aggregation and ex vivo platelet-dependent thrombus formation on immobilized collagen under low shear rate 500 s-1 indicating that free hemin is a strong activator for platelet-dependent thrombosis. Recently, we described that ACKR3 is a prominent inhibitory receptor of platelet activation. Specific ACKR3 agonists but not conventional antiplatelet compounds such as COX-1 inhibitor (indomethacin), ADP-receptor blocker (cangrelor), or PAR1 inhibitor (ML161) inhibit both hemin-dependent aggregation and thrombus formation. To further characterize the effect of hemin on platelet subpopulations we established a multi-color flow cytometry assay. We found that hemin induces procoagulant (CD42bpos/ PAC-1neg/ AnnexinVpos), aggregatory (CD42bpos / PAC-1pos / AnnexinVneg) and inflammatory (CD42bpos/ CXCR4pos/ACKR3pos/ AnnexinVpos) platelet subpopulations. Treatment with ACKR3 agonists significantly decrease the formation of procoagulant and ACKR3pos platelets in response to hemin. We conclude that hemin is a strong activator for the formation of procoagulant platelets and thrombus formation which is dependent on the function of ACKR3. Activation of ACKR3 through specific agonists may offer a therapeutic strategy to control vulnerability of atherosclerotic plaques in areas of IPH. Graphical abstractIntraplaque hemorrhages (IPH) results in hemolysis and liberation of iron containing heme and its oxidized metabolite hemin. Hemin activates platelets and has strong pro-thrombotic activity. Agonism of the atypical chemokine receptor 3 (ACKR3) inhibits hemin-induced platelet activation. O_FIG O_LINKSMALLFIG WIDTH=187 HEIGHT=200 SRC="FIGDIR/small/593847v2_ufig1.gif" ALT="Figure 1"> View larger version (78K): org.highwire.dtl.DTLVardef@1167f94org.highwire.dtl.DTLVardef@11ff58org.highwire.dtl.DTLVardef@192f098org.highwire.dtl.DTLVardef@1f1c568_HPS_FORMAT_FIGEXP M_FIG C_FIG

Platelet C-type lectin-like receptor 2 (CLEC-2) has been proposed as a potential anti-thrombotic target as genetic or antibody-mediated receptor deficiency prevents occlusive thrombus formation in mice. This occurs through interaction with an unknown ligand as the endogenous ligand podoplanin is not present in the vasculature. However, the CLEC-2-podoplanin interaction does have an important role in tumour metastasis. There are currently no methods to test potential human therapeutics targeting CLEC-2, such as antibodies, in vivo. We have therefore generated and characterised a humanised CLEC-2 mouse (hCLEC-2KI) and developed a novel monoclonal anti-human CLEC-2 antibody, HEL1, for in vivo testing. hCLEC-2KI mice were phenotypically normal and had comparable platelet glycoprotein receptor expression, activation and aggregation to wildtype platelets. hCLEC-2KI mice had both comparable bleeding and vessel occlusion times to WT mice. Challenging hCLEC-2KI mice with HEL1 or a second monoclonal anti-hCLEC-2 antibody, AYP1, resulted in transient thrombocytopenia as well as CLEC-2 depletion for more than 2 weeks but had no effect on haemostasis. This illustrates the power of the humanised CLEC-2 mouse model in evaluating novel therapeutics in vivo, including antibodies that target CLEC-2, as well as the limited effect on haemostasis when targeting CLEC-2.

Key PointsO_LIBispecific cis-acting platelet receptor inhibitors (CAPRIs) mediate hetero-clustering of the ITIM receptor G6B with ITAM receptors. C_LIO_LIG6B-GPVI and G6B-CD32A CAPRIs specifically inhibit collagen- and immune complex-induced thrombus formation, respectively. C_LI Platelets are highly reactive fragments of megakaryocytes that play a fundamental role in thrombosis and hemostasis. Predictably, all conventional anti-platelet therapies elicit bleeding, raising the question whether the thrombotic activity of platelets can be targeted separately. In this study, we describe a novel approach of inhibiting platelet activation through the use of bispecific single-chain variable fragments (bi-scFvs), termed cis-acting platelet receptor inhibitors (CAPRIs) that harness the immunoreceptor tyrosine-based inhibition motif (ITIM)-containing co-inhibitory receptor G6b-B (G6B) to suppress immunoreceptor tyrosine-based (ITAM)-containing receptor-mediated platelet activation. CAPRI-mediated hetero-clustering of G6B with either the ITAM-containing GPVI-FcR {gamma}-chain complex or Fc{gamma}RIIA (CD32A) inhibited collagen- or immune complex-induced platelet aggregation. G6B-GPVI CAPRIs strongly and specifically inhibited thrombus formation on collagen under arterial shear, whereas G6B-CD32A CAPRI strongly and specifically inhibited thrombus formation to heparin-induced thrombocytopenia, vaccine-induced thrombotic thrombocytopenia and antiphospholipid syndrome complexes on Von Willebrand Factor-coated surfaces and photochemical-injured endothelial cells under arterial shear. Our findings provide proof-of-concept that CAPRIs are highly effective at inhibiting ITAM receptor-mediated platelet activation, laying the foundation for a novel family of anti-thrombotic therapeutics with potentially improved efficacy and fewer bleeding outcomes compared with current anti-platelet therapies.

Neutrophil extracellular traps (NETs) are abundant in sepsis, and proposed NET-directed therapies in sepsis prevent their formation or accelerate degradation. Yet NETs are important for microbial entrapment, as NET digestion liberates pathogens and NET degradation products (NDPs) that deleteriously promote thrombosis and endothelial cell injury. We proposed an alternative strategy of NET-stabilization with the chemokine, platelet factor 4 (PF4, CXCL4), which we have shown enhances NET-mediated microbial entrapment. We now show that NET compaction by PF4 reduces their thrombogenicity. In vitro, we quantified plasma thrombin and fibrin generation by intact or degraded NETs and cell-free (cf) DNA fragments, and found that digested NETs and short DNA fragments were more thrombogenic than intact NETs and high molecular weight genomic DNA, respectively. PF4 reduced the thrombogenicity of digested NETs and DNA by interfering, in part, with contact pathway activation. In endothelial cell culture studies, short DNA fragments promoted von Willebrand factor release and tissue factor expression via a toll-like receptor 9-dependent mechanism. PF4 blocked these effects. Cxcl4-/- mice infused with cfDNA exhibited higher plasma thrombin anti-thrombin (TAT) levels compared to wild-type controls. Following challenge with bacterial lipopolysaccharide, Cxcl4-/- mice had similar elevations in plasma TAT and cfDNA, effects prevented by PF4 infusion. Thus, NET-stabilization by PF4 prevents the release of short fragments of cfDNA, limiting the activation of the contact coagulation pathway and reducing endothelial injury. These results support our hypothesis that NET-stabilization reduces pathologic sequelae in sepsis, an observation of potential clinical benefit. HIGHLIGHTSO_LIIn contrast to intact NETs, degraded NETs and cfDNA are prothrombotic and injure the endothelium. C_LIO_LIPF4 reduces the ability of degraded NETs and cfDNA to promote thrombosis and injure the endothelium. C_LI

It is challenging to develop anti-thrombotic therapeutics to treat or prevent pathological thrombus formation without increasing the risk of bleeding. Currently available anti-coagulant drugs render blood thinner irrespective of whether an inflammatory pro-thrombotic condition exists or whether clotting needs to occur because of traumatic vessel rupture. It is desirable to develop a drug that is active only under the oxidizing conditions present during inflammation. The blood protein von Willebrand factor (VWF) plays a key role in initiating blood clotting and it has been the target of anti-thrombotic therapies. It has been shown that oxidizing agents released during inflammation activate VWF by converting methionine residues within its domains to methionine sulfoxide. A previous study by us developed a method to computationally screen for drugs that inhibit VWF more strongly in the presence of oxidizing conditions. The computations suggested in particular a drug, lumacaftor, that could inhibit VWF function selectively in the presence of oxidized methionine residues. Here, we developed an enzyme-linked immunosorbent assay to test the effect of drugs on the ability of VWF to bind to the platelet surface receptor glycoprotein Ib comparing oxidizing and non-oxidizing conditions. The results indicate that lumacaftor may indeed have the desired properties of inhibiting VWF selectively when oxidized. Because of its simplicity, the assay provides a high-throughput method to efficiently screen multiple drugs against VWF in both its oxidized and unoxidized state.

Heparin-induced thrombocytopenia (HIT) is a severe immune-mediated prothrombotic disorder caused by antibodies reactive to complexes of platelet factor 4 and heparin. Platelets (PLTs) and their interaction with different immune cells contribute to prothrombotic conditions in HIT. However, the exact mechanisms and the role of different PLT subpopulations to this prothrombotic enviroment remain poorly understood. In this study, we observed that HIT patient antibodies (Abs) induce relevant changes in PLT phenotype, with the key features being increased P-Selectin expression and procoagulant phosphatidylserine (PS) externalization. Formation of procoagulant PLTs was dependent on engagement of PLT Fc-gamma-RIIA by HIT Abs and resulted in significant increase of thrombin generation on the PLT surface. Using an ex vivo thrombosis model and multi-parameter assessment of thrombus formation, we observed that HIT Ab-induced procoagulant PLTs propagated formation of large PLT aggregates, leukocyte recruitment and most importantly, fibrin network generation. These prothrombotic conditions were prevented via the upregulation of PLTs intracellular cAMP with Iloprost, a clinically approved prostacyclin analogue. Additionally, the functional relevance of high P-Selectin and PS levels on procoagulant PLTs was dissected. While inhibition of P-Selectin did not affect thrombus formation, the specific blockade of PS with Lactadherin prevented HIT Ab-mediated thrombin generation and most importantly procoagulant PLT-mediated thrombus formation ex vivo. Taken together, our findings indicate that procoagulant PLTs are critical mediators of prothrombotic conditions in HIT. Upregulation of cAMP with Iloprost or PS targeting specifc therapeutics could be a promising approach to prevent thromboembolic events in HIT patients. Key points- HIT immune complexes drive procoagulant platelet formation - Phosphatidylserine blockade prevents HIT antibody-induced thrombus formation

Background: Growing evidence suggests persistent thrombotic endothelial damage (together with elevated (fibrinaloid) microclot complexes (FMCs)) and immune dysfunction in the pathophysiology of Long COVID. Recently we proposed that there are different FMC phenotypes. Here we seek to determine the nature of these FMCs and aggregates in platelet-poor plasma (PPP) by using different markers, as well as thromboelastography (TEG) to assess for hypercoagulability of samples. Material and Methods: Whole-blood and PPP from control (n=19) and Long COVID (n=20) participants were assessed by thromboelastography. FMCs were quantified by imaging flow cytometry of Thioflavin-T (ThT)-stained PPP, 10X diluted PPP, and resuspended PPP pellets. The resuspended pellets were separately stained with a CD62P-PE antibody or Hoechst 33342 to label aggregates and FMCs containing amyloid, platelet, and nuclear material. ThT and CellMask Red were co-stained for confocal microscopy. ThT and myeloperoxidase (MPO), and ThT, Congo Red, and Hoechst were co-stained for fluorescence and polarized microscopy. Whole-blood smears were imaged by scanning electron microscopy (SEM). Results: Long COVID samples showed pronounced hypercoagulability in both whole blood and PPP, with shortened R, K and TMRTG and elevated alpha-angle and MRTG, but unchanged MA and TTG, indicating altered clotting kinetics. Persistence of this phenotype in PPP implicates soluble plasma constituents. ThT-positive FMCs were significantly increased in Long COVID across undiluted, diluted, and resuspended pellet samples; counts were processing-sensitive and a substantial ThT-positive population remained in the supernatant after centrifugation, indicating heterogeneity in density. Across probes, leukocyte material was the most abundant, then platelet material, and ThT-positive FMCs were the least abundant, with the three populations exhibiting unique morphology and occupying distinct size domains. Platelet-derived material was significantly elevated in Long COVID, whereas nuclear material was not. Co-stained samples subject to confocal, fluorescence, and polarized microscopy imaging showed that FMCs are heterogeneous, including events positive for ThT, CellMask, Hoechst, MPO, and Congo Red, and also a distinct subset of membrane-free, ThT-only events. Conclusion: In this Long COVID cohort, plasma is characterised by hypercoagulability and an increased burden of ThT-positive FMCs that are numerically minor relative to, and morphologically distinct from, aggregates and amyloidogenic FMCs marked with platelet- and leukocyte-derived material. The increased burden of platelet debris in PPP is likely indicative of persistent platelet activity. The existence of membrane-free, ThT-only FMCs, in addition to FMCs associated with cellular material, confirms an amyloid-dominated FMC population. Furthermore, positive Congo Red signal further confirms the amyloid nature of FMCs in PPP.

The tyrosine kinase BTK plays an important role in platelet function downstream of GPVI and CLEC2 receptors and has been proposed as a novel target to prevent thrombosis in patients that are at increased risk. However, current clinically approved BTK inhibitors have off target effects and are associated with an increased bleeding risk. In this study, we therefore explored whether BTK can be targeted for degradation in human platelets by using recently developed heterobifunctional molecules that employ the proteasomal system to break down BTK. Here we confirm that human platelets are highly susceptible to BTK degraders with the generic tyrosine kinase degrader TL12-186, and the BTK degraders DD-04-15 and DD-03-171 leading to breakdown of BTK and its closely related kinase TEC, an effect that was prevented by proteasomal inhibitors. Tandem Mass Tag proteomic analysis confirmed high selectivity with TL12-186 degrading BTK/TEC, FAK/PYK2 and FER, whereas DD-04-15 and DD-03-171 degraded BTK/TEC only. GPVI-mediated platelet integrin IIb{beta}3 activation, P-selectin expression, and phosphatidyl-serine exposure were largely impaired upon BTK/TEC degradation, with PAR-1-mediated responses left intact. This is the first study to demonstrate that chemical protein degraders can be successfully employed in anucleate human platelets to modulate their function.