4D intravital imaging identifies platelets as the predominant cellular procoagulant surface in a mouse hemostasis model.

Interplay between platelets, coagulation factors, endothelial cells (ECs) and fibrinolytic factors is necessary for effective hemostatic plug formation. This study describes a four-dimensional (4D) imaging platform to visualize and quantify hemostatic plug components in mice with high spatiotemporal resolution. Fibrin accumulation following laser-induced vascular injury was observed at the platelet plug-EC interface, controlled by the antagonistic balance between fibrin generation and breakdown. We observed less fibrin accumulation in mice expressing low levels of tissue factor (TFlow) or F12-/- mice compared to controls, whereas increased fibrin accumulation, including on the vasculature adjacent to the platelet plug, was observed in plasminogen-deficient mice or wild-type mice treated with tranexamic acid (TXA). Phosphatidylserine (PS), a membrane lipid critical for the assembly of coagulation factors, was first detected at the platelet plug-EC interface, followed by exposure across the endothelium. Impaired PS exposure resulted in a significant reduction in fibrin accumulation in cyclophilin D-/- mice. Adoptive transfer studies demonstrated a key role for PS exposure on platelets, and to a lesser degree on ECs, in fibrin accumulation during hemostatic plug formation. Together, these studies suggest that (1) platelets are the functionally dominant procoagulant cellular surface, and (2) plasmin is critical for limiting fibrin accumulation at the site of a forming hemostatic plug.

Mice expressing nonpolymerizable fibrinogen have reduced arterial and venous thrombosis with preserved hemostasis.

ABSTRACT: Elevated circulating fibrinogen levels correlate with increased risk for both cardiovascular and venous thromboembolic diseases. In vitro studies show that formation of a highly dense fibrin matrix is a major determinant of clot structure and stability. Here, we analyzed the impact of nonpolymerizable fibrinogen on arterial and venous thrombosis as well as hemostasis in vivo using FgaEK mice that express normal levels of a fibrinogen that cannot be cleaved by thrombin. In a model of carotid artery thrombosis, FgaWT/EK and FgaEK/EK mice were protected from occlusion with 4% ferric chloride (FeCl3) challenges compared with wild-type (FgaWT/WT) mice, but this protection was lost, with injuries driven by higher concentrations of FeCl3. In contrast, fibrinogen-deficient (Fga-/-) mice showed no evidence of occlusion, even with high-concentration FeCl3 challenge. Fibrinogen-dependent platelet aggregation and intraplatelet fibrinogen content were similar in FgaWT/WT, FgaWT/EK, and FgaEK/EK mice, consistent with preserved fibrinogen-platelet interactions that support arterial thrombosis with severe challenge. In an inferior vena cava stasis model of venous thrombosis, FgaEK/EK mice had near complete protection from thrombus formation. FgaWT/EK mice also displayed reduced thrombus incidence and a significant reduction in thrombus mass relative to FgaWT/WT mice after inferior vena cava stasis, suggesting that partial expression of nonpolymerizable fibrinogen was sufficient for conferring protection. Notably, FgaWT/EK and FgaEK/EK mice had preserved hemostasis in multiple models as well as normal wound healing times after skin incision, unlike Fga-/- mice that displayed significant bleeding and delayed healing. These findings indicate that a nonpolymerizable fibrinogen variant can significantly suppress occlusive thrombosis while preserving hemostatic potential in vivo.

4D intravital imaging studies identify platelets as the predominant cellular procoagulant surface in a mouse model of hemostasis.

Interplay between platelets, coagulation/fibrinolytic factors, and endothelial cells (ECs) is necessary for effective hemostatic plug formation. This study describes a novel four-dimensional (4D) imaging platform to visualize and quantify hemostatic plug components with high spatiotemporal resolution. Fibrin accumulation following laser-induced endothelial ablation was observed at the EC-platelet plug interface, controlled by the antagonistic balance between fibrin generation and breakdown. Phosphatidylserine (PS) was first detected in close physical proximity to the fibrin ring, followed by exposure across the endothelium. Impaired PS exposure in cyclophilinD -/- mice resulted in a significant reduction in fibrin accumulation. Adoptive transfer and inhibitor studies demonstrated a key role for platelets, but not ECs, in fibrin generation during hemostatic plug formation. Inhibition of fibrinolysis with tranexamic acid (TXA) led to increased fibrin accumulation in WT mice, but not in cyclophilinD -/- mice or WT mice treated with antiplatelet drugs. These studies implicate platelets as the functionally dominant procoagulant surface during hemostatic plug formation. In addition, they suggest that impaired fibrin formation due to reduced platelet procoagulant activity is not reversed by TXA treatment.

Loss of P2Y1 receptor desensitization does not impact hemostasis or thrombosis despite increased platelet reactivity in vitro.

BACKGROUND: The hemostatic plug formation at sites of vascular injury is strongly dependent on rapid platelet activation and integrin-mediated adhesion and aggregation. However, to prevent thrombotic complications, platelet aggregate formation must be a self-limiting process. The second-wave mediator adenosine diphosphate (ADP) activates platelets via Gq-coupled P2Y1 and Gi-coupled P2Y12 receptors. After ADP exposure, the P2Y1 receptor undergoes rapid phosphorylation-induced desensitization, a negative feedback mechanism believed to be critical for limiting thrombus growth. OBJECTIVE: The objective of this study was to examine the role of rapid P2Y1 receptor desensitization on platelet function and thrombus formation in vivo. METHODS: We analyzed a novel knock-in mouse strain expressing a P2Y1 receptor variant that cannot be phosphorylated beyond residue 340 (P2Y1340-0P), thereby preventing the desensitization of the receptor. RESULTS: P2Y1340-0P mice followed a Mendelian inheritance pattern, and peripheral platelet counts were comparable between P2Y1340-0P/340-0P and control mice. In vitro, P2Y1340-0P/340-0P platelets were hyperreactive to ADP, showed a robust activation response to the P2Y1 receptor-selective agonist, MRS2365, and did not desensitize in response to repeated ADP challenge. We observed increased calcium mobilization, protein kinase C substrate phosphorylation, alpha granule release, activation of the small GTPase Rap1, and integrin inside-out activation/aggregation. This hyperreactivity, however, did not lead to increased platelet adhesion or excessive plug formation under physiological shear conditions. CONCLUSION: Our studies demonstrate that receptor phosphorylation at the C-terminus is critical for P2Y1 receptor desensitization in platelets and that impaired desensitization leads to increased P2Y1 receptor signaling in vitro. Surprisingly, desensitization of the P2Y1 receptor is not required for limiting platelet adhesion/aggregation at sites of vascular injury, likely because ADP is degraded quickly or washed away in the bloodstream.

Decreased Platelet Reactivity and Function in a Mouse Model of Human Pancreatic Cancer.

Cancer patients have increased thrombosis and bleeding compared with the general population. Cancer is associated with activation of both platelets and coagulation. Mouse models have been used to study the dysregulation of platelets and coagulation in cancer. We established a mouse model of pancreatic cancer in which tissue factor-expressing human pancreatic tumors (BxPC-3) are grown in nude mice. Tumor-bearing mice have an activated coagulation system and increased venous thrombosis compared to control mice. We also showed that tumor-derived, tissue factor-positive extracellular vesicles activated platelets ex vivo and in vivo. In this study, we determined the effect of tumors on a platelet-dependent arterial thrombosis model. Unexpectedly, we observed significantly reduced carotid artery thrombosis in tumor-bearing mice compared to controls. In addition, we observed significantly increased tail bleeding in tumor-bearing mice compared to controls. These results suggested that the presence of the tumor affected platelets. Indeed, tumor-bearing mice exhibited a significant decrease in platelet count and an increase in mean platelet volume and percentage of reticulated platelets, findings that are consistent with increased platelet turnover. Levels of the platelet activation marker platelet factor 4 were also increased in tumor-bearing mice. We also observed decreased platelet receptor expression in tumor-bearing mice and reduced levels of active αIIb/ß3 integrin in response to PAR4 agonist peptide and convulxin in platelets from tumor-bearing mice compared with platelets from control mice. In summary, our study suggests that in tumor-bearing mice there is chronic platelet activation, leading to thrombocytopenia, decreased receptor expression, and impaired platelet adhesive function.

Both G protein-coupled and immunoreceptor tyrosine-based activation motif receptors mediate venous thrombosis in mice.

Platelets are critical in hemostasis and a major contributor to arterial thrombosis (AT). (Pre)clinical studies suggest platelets also contribute to venous thrombosis (VT), but the mechanisms are largely unknown. We hypothesized that in VT, platelets use signaling machinery distinct from AT. Here we aimed to characterize the contributions of platelet G protein-coupled (GPCR) and immunoreceptor tyrosine-based activation motif (ITAM) receptor signaling to VT. Wild-type (WT) and transgenic mice were treated with inhibitors to selectively inhibit platelet-signaling pathways: ITAM-CLEC2 (Clec2mKO), glycoprotein VI (JAQ1 antibody), and Bruton's tyrosine kinase (ibrutinib); GPCR-cyclooxygenase 1 (aspirin); and P2Y12 (clopidogrel). VT was induced by inferior vena cava stenosis. Thrombin generation in platelet-rich plasma and whole-blood clot formation were studied ex vivo. Intravital microscopy was used to study platelet-leukocyte interactions after flow restriction. Thrombus weights were reduced in WT mice treated with high-dose aspirin + clopidogrel (dual antiplatelet therapy [DAPT]) but not in mice treated with either inhibitor alone or low-dose DAPT. Similarly, thrombus weights were reduced in mice with impaired ITAM signaling (Clec2mKO + JAQ1; WT + ibrutinib) but not in Clec2mKO or WT + JAQ1 mice. Both aspirin and clopidogrel, but not ibrutinib, protected mice from FeCl3-induced AT. Thrombin generation and clot formation were normal in blood from high-dose DAPT- or ibrutinib-treated mice; however, platelet adhesion and platelet-neutrophil aggregate formation at the vein wall were reduced in mice treated with high-dose DAPT or ibrutinib. In summary, VT initiation requires platelet activation via GPCRs and ITAM receptors. Strong inhibition of either signaling pathway reduces VT in mice.

Fibrin(ogen) engagement of S. aureus promotes the host antimicrobial response and suppression of microbe dissemination following peritoneal infection.

The blood-clotting protein fibrin(ogen) plays a critical role in host defense against invading pathogens, particularly against peritoneal infection by the Gram-positive microbe Staphylococcus aureus. Here, we tested the hypothesis that direct binding between fibrin(ogen) and S. aureus is a component of the primary host antimicrobial response mechanism and prevention of secondary microbe dissemination from the peritoneal cavity. To establish a model system, we showed that fibrinogen isolated from FibγΔ5 mice, which express a mutant form lacking the final 5 amino acids of the fibrinogen γ chain (termed fibrinogenγΔ5), did not support S. aureus adherence when immobilized and clumping when in suspension. In contrast, purified wildtype fibrinogen supported robust adhesion and clumping that was largely dependent on S. aureus expression of the receptor clumping factor A (ClfA). Following peritoneal infection with S. aureus USA300, FibγΔ5 mice displayed worse survival compared to WT mice coupled to reduced bacterial killing within the peritoneal cavity and increased dissemination of the microbes into circulation and distant organs. The failure of acute bacterial killing, but not enhanced dissemination, was partially recapitulated by mice infected with S. aureus USA300 lacking ClfA. Fibrin polymer formation and coagulation transglutaminase Factor XIII each contributed to killing of the microbes within the peritoneal cavity, but only elimination of polymer formation enhanced systemic dissemination. Host macrophage depletion or selective elimination of the fibrin(ogen) ß2-integrin binding motif both compromised local bacterial killing and enhanced S. aureus systemic dissemination, suggesting fibrin polymer formation in and of itself was not sufficient to retain S. aureus within the peritoneal cavity. Collectively, these findings suggest that following peritoneal infection, the binding of S. aureus to stabilized fibrin matrices promotes a local, macrophage-mediated antimicrobial response essential for prevention of microbe dissemination and downstream host mortality.

Hypofibrinogenemia with preserved hemostasis and protection from thrombosis in mice with an Fga truncation mutation.

Genetic variants within the fibrinogen Aα chain encoding the αC-region commonly result in hypodysfibrinogenemia in patients. However, the (patho)physiological consequences and underlying mechanisms of such mutations remain undefined. Here, we generated Fga270 mice carrying a premature termination codon within the Fga gene at residue 271. The Fga270 mutation was compatible with Mendelian inheritance for offspring of heterozygous crosses. Adult Fga270/270 mice were hypofibrinogenemic with ∼10% plasma fibrinogen levels relative to FgaWT/WT mice, linked to 90% reduction in hepatic Fga messenger RNA (mRNA) because of nonsense-mediated decay of the mutant mRNA. Fga270/270 mice had preserved hemostatic potential in vitro and in vivo in models of tail bleeding and laser-induced saphenous vein injury, whereas Fga-/- mice had continuous bleeding. Platelets from FgaWT/WT and Fga270/270 mice displayed comparable initial aggregation following adenosine 5'-diphosphate stimulation, but Fga270/270 platelets quickly disaggregated. Despite ∼10% plasma fibrinogen, the fibrinogen level in Fga270/270 platelets was ∼30% of FgaWT/WT platelets with a compensatory increase in fibronectin. Notably, Fga270/270 mice showed complete protection from thrombosis in the inferior vena cava stasis model. In a model of Staphylococcus aureus peritonitis, Fga270/270 mice supported local, fibrinogen-mediated bacterial clearance and host survival comparable to FgaWT/WT, unlike Fga-/- mice. Decreasing the normal fibrinogen levels to ∼10% with small interfering RNA in mice also provided significant protection from venous thrombosis without compromising hemostatic potential and antimicrobial function. These findings both reveal novel molecular mechanisms underpinning fibrinogen αC-region truncation mutations and highlight the concept that selective fibrinogen reduction may be efficacious for limiting thrombosis while preserving hemostatic and immune protective functions.

Novel Mouse Model for Studying Hemostatic Function of Human Platelets.

OBJECTIVE: Platelets are critical to the formation of a hemostatic plug and the pathogenesis of atherothrombosis. Preclinical animal models, especially the mouse, provide an important platform to assess the efficacy and safety of antiplatelet drugs. However, these studies are limited by inherent differences between human and mouse platelets and the species-selectivity of many drugs. To circumvent these limitations, we developed a new protocol for the adoptive transfer of human platelets into thrombocytopenic nonobese diabetic/severe combined immune deficiency mice, that is, a model where all endogenous platelets are replaced by human platelets in mice accepting xenogeneic tissues. Approach and Results: To demonstrate the power of this new model, we visualized and quantified hemostatic plug formation and stability by intravital spinning disk confocal microscopy following laser ablation injury to the saphenous vein. Integrin αIIbß3-dependent hemostatic platelet plug formation was achieved within ≈30 seconds after laser ablation injury in humanized platelet mice. Pretreatment of mice with standard dual antiplatelet therapy (Aspirin+Ticagrelor) or PAR1 inhibitor, L-003959712 (an analog of vorapaxar), mildly prolonged the bleeding time and significantly reduced platelet adhesion to the site of injury. Consistent with findings from clinical trials, inhibition of PAR1 in combination with dual antiplatelet therapy markedly prolonged bleeding time in humanized platelet mice. CONCLUSIONS: We propose that this novel mouse model will provide a robust platform to test and predict the safety and efficacy of experimental antiplatelet drugs and to characterize the hemostatic function of synthetic, stored and patient platelets.

Talin-1 is the principal platelet Rap1 effector of integrin activation.

Ras-related protein 1 (Rap1) is a major convergence point of the platelet-signaling pathways that result in talin-1 binding to the integrin ß cytoplasmic domain and consequent integrin activation, platelet aggregation, and effective hemostasis. The nature of the connection between Rap1 and talin-1 in integrin activation is an important remaining gap in our understanding of this process. Previous work identified a low-affinity Rap1-binding site in the talin-1 F0 domain that makes a small contribution to integrin activation in platelets. We recently identified an additional Rap1-binding site in the talin-1 F1 domain that makes a greater contribution than F0 in model systems. Here we generated mice bearing point mutations, which block Rap1 binding without affecting talin-1 expression, in either the talin-1 F1 domain (R118E) alone, which were viable, or in both the F0 and F1 domains (R35E,R118E), which were embryonic lethal. Loss of the Rap1-talin-1 F1 interaction in platelets markedly decreases talin-1-mediated activation of platelet ß1- and ß3-integrins. Integrin activation and platelet aggregation in mice whose platelets express only talin-1(R35E, R118E) are even more impaired, resembling the defect seen in platelets lacking both Rap1a and Rap1b. Although Rap1 is important in thrombopoiesis, platelet secretion, and surface exposure of phosphatidylserine, loss of the Rap1-talin-1 interaction in talin-1(R35E, R118E) platelets had little effect on these processes. These findings show that talin-1 is the principal direct effector of Rap1 GTPases that regulates platelet integrin activation in hemostasis.

Rap1 binding to the talin 1 F0 domain makes a minimal contribution to murine platelet GPIIb-IIIa activation.

Activation of platelet glycoprotein IIb-IIIa (GPIIb-IIIa; integrin αIIbß3) leads to high-affinity fibrinogen binding and platelet aggregation during hemostasis. Whereas GTP-bound Rap1 GTPase promotes talin 1 binding to the ß3 cytoplasmic domain to activate platelet GPIIb-IIIa, the Rap1 effector that regulates talin association with ß3 in platelets is unknown. Rap1 binding to the talin 1 F0 subdomain was proposed to forge the talin 1-Rap1 link in platelets. Here, we report a talin 1 point mutant (R35E) that significantly reduces Rap1 affinity without a significant effect on its structure or expression. Talin 1 head domain (THD) (R35E) was of similar potency to wild-type THD in activating αIIbß3 in Chinese hamster ovary cells. Coexpression with activated Rap1b increased activation, and coexpression with Rap1GAP1 reduced activation caused by transfection of wild-type THD or THD(R35E). Furthermore, platelets from Tln1R35E/R35E mice showed similar GPIIb-IIIa activation to those from wild-type littermates in response to multiple agonists. Tln1R35E/R35E platelets exhibited slightly reduced platelet aggregation in response to low doses of agonists; however, there was not a significant hemostatic defect, as judged by tail bleeding times. Thus, the Rap1-talin 1 F0 interaction has little effect on platelet GPIIb-IIIa activation and hemostasis and cannot account for the dramatic effects of loss of Rap1 activity on these platelet functions.

Functional redundancy between RAP1 isoforms in murine platelet production and function.

RAP GTPases, important regulators of cellular adhesion, are abundant signaling molecules in the platelet/megakaryocytic lineage. However, mice lacking the predominant isoform, RAP1B, display a partial platelet integrin activation defect and have a normal platelet count, suggesting the existence of a RAP1-independent pathway to integrin activation in platelets and a negligible role for RAP GTPases in megakaryocyte biology. To determine the importance of individual RAP isoforms on platelet production and on platelet activation at sites of mechanical injury or vascular leakage, we generated mice with megakaryocyte-specific deletion (mKO) of Rap1a and/or Rap1b Interestingly, Rap1a/b-mKO mice displayed a marked macrothrombocytopenia due to impaired proplatelet formation by megakaryocytes. In platelets, RAP isoforms had redundant and isoform-specific functions. Deletion of RAP1B, but not RAP1A, significantly reduced α-granule secretion and activation of the cytoskeleton regulator RAC1. Both isoforms significantly contributed to thromboxane A2 generation and the inside-out activation of platelet integrins. Combined deficiency of RAP1A and RAP1B markedly impaired platelet aggregation, spreading, and clot retraction. Consistently, thrombus formation in physiological flow conditions was abolished in Rap1a/b-mKO, but not Rap1a-mKO or Rap1b-mKO, platelets. Rap1a/b-mKO mice were strongly protected from experimental thrombosis and exhibited a severe defect in hemostasis after mechanical injury. Surprisingly, Rap1a/b-mKO platelets were indistinguishable from controls in their ability to prevent blood-lymphatic mixing during development and hemorrhage at sites of inflammation. In summary, our studies demonstrate an essential role for RAP1 signaling in platelet integrin activation and a critical role in platelet production. Although important for hemostatic/thrombotic plug formation, platelet RAP1 signaling is dispensable for vascular integrity during development and inflammation.

Protein kinase C signaling dysfunction in von Willebrand disease (p.V1316M) type 2B platelets.

von Willebrand disease (VWD) type 2B is characterized by gain-of-function mutations in von Willebrand factor (VWF), enhancing its binding affinity for the platelet receptor glycoprotein (GP)Ibα. VWD type 2B patients display a bleeding tendency associated with loss of high-molecular-weight VWF multimers and variable thrombocytopenia. We recently demonstrated that a marked defect in agonist-induced activation of the small GTPase, Rap1, and integrin αIIbß3 in VWD (p.V1316M) type 2B platelets also contributes to the bleeding tendency. Here, we investigated the molecular mechanisms underlying impaired platelet Rap1 signaling in this disease. Two distinct pathways contribute to Rap1 activation in platelets: rapid activation mediated by the calcium-sensing guanine nucleotide exchange factor CalDAG-GEF-I (CDGI) and sustained activation that is dependent on signaling by protein kinase C (PKC) and the adenosine 5'-diphosphate receptor P2Y12. To investigate which Rap1 signaling pathway is affected, we expressed VWF/p.V1316M by hydrodynamic gene transfer in wild-type and Caldaggef1-/- mice. Using αIIbß3 integrin activation as a read-out, we demonstrate that platelet dysfunction in VWD (p.V1316M) type 2B affects PKC-mediated, but not CDGI-mediated, activation of Rap1. Consistently, we observed decreased PKC substrate phosphorylation and impaired granule release in stimulated VWD type 2B platelets. Interestingly, the defect in PKC signaling was caused by a significant increase in baseline PKC substrate phosphorylation in circulating VWD (p.V1316M) type 2B platelets, suggesting that the VWF-GPIbα interaction leads to preactivation and exhaustion of the PKC pathway. Consistent with PKC preactivation, VWD (p.V1316M) type 2B mice also exhibited marked shedding of platelet GPIbα. In summary, our studies identify altered PKC signaling as the underlying cause of platelet hypofunction in p.V1316M-associated VWD type 2B.

Identification of two novel mutations in RASGRP2 affecting platelet CalDAG-GEFI expression and function in patients with bleeding diathesis.

The RASGRP2 gene encodes the Ca2+ and DAG-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), which plays a key role in integrin activation in platelets and neutrophils. We here report two new RASGRP2 variants associated with platelet dysfunction and bleeding in patients. The homozygous patients had normal platelet and neutrophil counts and morphology. Platelet phenotyping showed: prolonged PFA-100 closure times; normal expression of major glycoprotein receptors; severely reduced platelet aggregation response to ADP and collagen (both patients); aggregation response to PAR1 and arachidonic acid markedly impaired in one patient; PMA-induced aggregation unaffected; platelet secretion, clot retraction, and spreading minimally affected. Genetic analysis identified two new homozygous variants in RASGRP2: c.706C>T (p.Q236X) and c.887G>A (p.C296Y). In both patients, CalDAG-GEFI protein was not detectable in platelet lysates, and platelet αIIbß3 activation, as assessed by fibrinogen binding, was greatly impaired in response to all agonists except PMA. Patient neutrophils showed normal integrin expression, but impaired Mn2+-induced fibrinogen binding. In summary, we have identified two new RASGRP2 mutations that can be added to this rapidly growing form of inherited platelet function disorder.

Marked bleeding diathesis in patients with platelet dysfunction due to a novel mutation in RASGRP2, encoding CalDAG-GEFI (p.Gly305Asp).

Congenital platelet function disorders are often the result of defects in critical signal transduction pathways required for platelet adhesion and clot formation. Mutations affecting RASGRP2, the gene encoding the Rap GTPase activator, CalDAG-GEFI, give rise to a novel, and rare, group of platelet signal transduction abnormalities. We here report platelet function studies for two brothers (P1 and P2) expressing a novel variant of RASGRP2, CalDAG-GEFI(p.Gly305Asp). P1 and P2 have a lifelong history of bleeding with severe epistaxis successfully treated with platelet transfusions or rFVIIa. Other bleedings include extended hemorrhage from minor wounds. Platelet counts and plasma coagulation were normal, as was αIIbß3 and GPIb expression on the platelet surface. Aggregation of patients' platelets was significantly impaired in response to select agonists including ADP, epinephrine, collagen, and calcium ionophore A23187. Integrin αIIbß3 activation and granule release were also impaired. CalDAG-GEFI protein expression was markedly reduced but not absent. Homology modeling places the Gly305Asp substitution at the GEF-Rap1 interface, suggesting that the mutant protein has very limited catalytic activity. In summary, we here describe a novel mutation in RASGRP2 that affects both expression and function of CalDAG-GEFI and that causes impaired platelet adhesive function and significant bleeding in humans.

Deletion of the Arp2/3 complex in megakaryocytes leads to microthrombocytopenia in mice.

Actin reorganization regulates key processes in platelet activation. Here we examined the role of the Arp2/3 complex, an essential component in actin filament branching, in platelet function. The Arpc2 gene, encoding the p34 subunit of the Arp2/3 complex, was deleted in the megakaryocyte lineage (Arpc2fl/flPF4-Cre). Deletion of the Arp2/3 complex resulted in marked microthrombocytopenia in mice, caused by premature platelet release into the bone marrow compartment and impaired platelet survival in circulation. Arpc2fl/flPF4-Cre platelets exhibited alterations in their actin cytoskeleton and their peripheral microtubule coil. Thrombocytopenia was alleviated following clodronate liposome-induced macrophage depletion in Arpc2fl/flPF4-Cre mice. Arpc2fl/flPF4-Cre platelets failed to spread and showed a mild defect in integrin activation and aggregation. However, no significant differences in hemostasis or thrombosis were observed between Arpc2fl/flPF4-Cre and control mice. Thus, Arp2/3 is critical for platelet homeostasis but plays only a minor role for vascular hemostasis.

Phenotype analysis and clinical management in a large family with a novel truncating mutation in RASGRP2, the CalDAG-GEFI encoding gene.

BACKGROUND: Genetic variants in the RASGRP2 gene encoding calcium and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) represent a new inherited bleeding disorder linked to major defects of platelet aggregation and activation of αIIbß3 integrin. They are of major interest as CalDAG-GEFI is receiving attention as a potential target for antiplatelet therapy for prevention and treatment of cardiovascular disorders including arterial thrombosis and atherosclerosis. OBJECTIVES: To better understand the phenotypical and clinical profiles of patients with CalDAG-GEFI deficiency. PATIENTS: We report a five-generation family with a novel truncating CalDAG-GEFI mutation detailing clinical management and phenotypic variability. RESULTS: Patients IV.6 & IV.4 manifested with episodes of serious mucocutanous bleeding or bleeding after surgery not responding to platelet transfusion but responding well to recombinant Factor VIIa infusions. Their blood counts and coagulation parameters were normal but platelet aggregation to ADP and collagen was defective. Further work-up confirmed normal levels of αIIb and ß3 in their platelets but decreased αIIbß3 function. DNA analysis by whole exome sequencing within the BRIDGE-BPD consortium (Cambridge, UK), allowed us to highlight a homozygous c.1490delT predicted to give rise to a p.F497Sfs*22 truncating mutation near to the C-terminal domain of CalDAG-GEFI. Sanger sequencing confirmed that both patients were homozygous for the c.1490delT and 3 out of 4 close family members were heterozygous. CONCLUSIONS: A long-term prospective study is warranted for full clinical exploration of CalDAG-GEFI to understand the bleeding phenotyes and their management.

The Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) Binder Rasa3 Regulates Phosphoinositide 3-kinase (PI3K)-dependent Integrin αIIbß3 Outside-in Signaling.

The class I PI3K family of lipid kinases plays an important role in integrin αIIbß3 function, thereby supporting thrombus growth and consolidation. Here, we identify Ras/Rap1GAP Rasa3 (GAP1IP4BP) as a major phosphatidylinositol 3,4,5-trisphosphate-binding protein in human platelets and a key regulator of integrin αIIbß3 outside-in signaling. We demonstrate that cytosolic Rasa3 translocates to the plasma membrane in a PI3K-dependent manner upon activation of human platelets. Expression of wild-type Rasa3 in integrin αIIbß3-expressing CHO cells blocked Rap1 activity and integrin αIIbß3-mediated spreading on fibrinogen. In contrast, Rap1GAP-deficient (P489V) and Ras/Rap1GAP-deficient (R371Q) Rasa3 had no effect. We furthermore show that two Rasa3 mutants (H794L and G125V), which are expressed in different mouse models of thrombocytopenia, lack both Ras and Rap1GAP activity and do not affect integrin αIIbß3-mediated spreading of CHO cells on fibrinogen. Platelets from thrombocytopenic mice expressing GAP-deficient Rasa3 (H794L) show increased spreading on fibrinogen, which in contrast to wild-type platelets is insensitive to PI3K inhibitors. Together, these results support an important role for Rasa3 in PI3K-dependent integrin αIIbß3-mediated outside-in signaling and cell spreading.

Platelet clearance via shear-induced unfolding of a membrane mechanoreceptor.

Mechanisms by which blood cells sense shear stress are poorly characterized. In platelets, glycoprotein (GP)Ib-IX receptor complex has been long suggested to be a shear sensor and receptor. Recently, a relatively unstable and mechanosensitive domain in the GPIbα subunit of GPIb-IX was identified. Here we show that binding of its ligand, von Willebrand factor, under physiological shear stress induces unfolding of this mechanosensory domain (MSD) on the platelet surface. The unfolded MSD, particularly the juxtamembrane 'Trigger' sequence therein, leads to intracellular signalling and rapid platelet clearance. These results illustrate the initial molecular event underlying platelet shear sensing and provide a mechanism linking GPIb-IX to platelet clearance. Our results have implications on the mechanism of platelet activation, and on the pathophysiology of von Willebrand disease and related thrombocytopenic disorders. The mechanosensation via receptor unfolding may be applicable for many other cell adhesion receptors.

Mice Expressing Low Levels of CalDAG-GEFI Exhibit Markedly Impaired Platelet Activation With Minor Impact on Hemostasis.

OBJECTIVE: The tight regulation of platelet adhesiveness, mediated by the αIIbß3 integrin, is critical for hemostasis and prevention of thrombosis. We recently demonstrated that integrin affinity in platelets is controlled by the guanine nucleotide exchange factor, CalDAG-GEFI (CD-GEFI), and its target, RAP1. In this study, we investigated whether low-level expression of CD-GEFI leads to protection from thrombosis without pathological bleeding in mice. APPROACH AND RESULTS: Cdg1(low) mice were generated by knockin of human CD-GEFI cDNA into the mouse Cdg1 locus. CD-GEFI expression in platelets from Cdg1(low) mice was reduced by ≈90% when compared with controls. Activation of RAP1 and αIIbß3 was abolished at low agonist concentrations and partially inhibited at high agonist concentrations in Cdg1(low) platelets. Consistently, the aggregation response of Cdg1(low) platelets was weaker than that of wild-type platelets, but more efficient than that observed in Cdg1(-/-) platelets. Importantly, Cdg1(low) mice were strongly protected from arterial and immune complex-mediated thrombosis, with only minimal impact on primary hemostasis. CONCLUSIONS: Together, our studies suggest the partial inhibition of CD-GEFI function as a powerful new approach to safely prevent thrombotic complications.