Platelet-collagen Interactions Research Articles

Von Willebrand Factor Collagen-Binding Activity and Von Willebrand Factor-Mediated Platelet Adhesion in Patients with Coronary Artery Disease

In this study, we investigated von Willebrand factor (VWF)-related parameters in 30 patients with stable coronary artery disease (CAD) and 50 patients without CAD. In both groups, the following were measured: the VWF antigen level (VWF:Ag); the VWF ristocetin cofactor activity (VWF:RCo); the VWF collagen-binding activity (VWF:CB); and VWF-mediated platelet adhesion. Platelet adhesion was measured in whole blood at a shear rate of 1300 s−1 using a microfluidic chamber with a collagen-coated surface. VWF:Ag and VWF:RCo were found to be the same in both groups of patients. However, VWF:CB was found to be lower in patients with CAD compared with patients without CAD, with values of 106.7% (82.1; 131.6) and 160.4% (112.5; 218.1), respectively (p < 0.001). The decrease in platelet adhesion after GPIb inhibition was more pronounced in patients with CAD compared with patients of the control group, with recorded values of 76.0% (60.6; 82.1) and 29.3% (0.0; 60.4), respectively (p < 0.001). After adjusting for traditional risk factors, the odds ratio for CAD was found to be 0.98 (95% CI, 0.97–0.99; p = 0.011) per 1% increase in VWF:CB activity, and 1.06 (95% CI, 1.03–1.09; p < 0.001) per 1% decrease in GPIb-mediated platelet adhesion. The findings presented in this paper indicate a possible critical role played by complex VWF–collagen-platelet interactions in the development of CAD.

LAIR-1 and PECAM-1 function via the same signaling pathway to inhibit GPVI-mediated platelet activation

BackgroundInhibition of platelet responsiveness is important for controlling thrombosis. It is well established that platelet endothelial cell adhesion molecule-1 (PECAM-1) serves as a physiological negative regulator of platelet-collagen interactions. We recently demonstrated that leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is a negative regulator of platelet production and reactivity. It is however not known if LAIR-1 and PECAM-1 function in the same or different inhibitory pathways. ObjectivesIn this study, we investigated the role of LAIR-1 alongside PECAM-1 in megakaryocyte development and platelet production and determined the functional redundancy through characterization of a LAIR-1/PECAM-1 double knockout (DKO) mouse model. MethodsLAIR-1 and PECAM-1 expression in megakaryocytes were evaluated by western blotting. Megakaryocyte ploidy and proplatelet formation were evaluated by flow cytometry and fluorescent microscopy. Platelet function and signalling were compared in wild-type, LAIR-1−/−, PECAM-1−/− and DKO mice using aggregometry, flow cytometry and western blotting. Thrombosis was evaluated using the FeCl3 carotid artery model. ResultsWe show that LAIR-1/PECAM-1 DKO mice exhibit a 17% increase in platelet count. Bone marrow-derived megakaryocytes from all 3 mouse models had normal ploidy in vitro, suggesting that neither LAIR-1 nor PECAM-1 regulates megakaryocyte development. Furthermore, relative to wild-type platelets, platelets derived from LAIR-1, PECAM-1, and DKO mice were equally hyperresponsive to collagen in vitro, indicating that LAIR-1 and PECAM-1 participate in the same inhibitory pathway. Interestingly, DKO mice exhibited normal thrombus formation in vivo due to DKO mouse platelets lacking the enhanced Src family kinase activation previously shown in platelets from LAIR-1-deficient mice. ConclusionFindings from this study reveal that LAIR-1 and PECAM-1 act to inhibit GPVI-mediated platelet activation via the same signaling pathway. Mice lacking LAIR-1 and PECAM-1 do not however exhibit an increase in thrombus formation despite minor increase in platelet count and reactivity to collagen. This study adds to the growing evidence that immunoreceptor tyrosine-based inhibition motif–containing receptors are important regulators of platelet count and function.

Pharmacological Inhibition of Glycoprotein VI- and Integrin α2β1-Induced Thrombus Formation Modulated by the Collagen Type.

In secondary cardiovascular disease prevention, treatments blocking platelet-derived secondary mediators pose a risk of bleeding. Pharmacological interference of the interaction of platelets with exposed vascular collagens is an attractive alternative, with clinical trials ongoing. Antagonists of the collagen receptors, glycoprotein VI (GPVI), and integrin α2β1, include recombinant GPVI-Fc dimer construct Revacept, 9O12 mAb based on the GPVI-blocking reagent Glenzocimab, Syk tyrosine-kinase inhibitor PRT-060318, and anti-α2β1 mAb 6F1. No direct comparison has been made of the antithrombic potential of these drugs. Using a multiparameter whole-blood microfluidic assay, we compared the effects of Revacept, 9O12-Fab, PRT-060318, or 6F1 mAb intervention with vascular collagens and collagen-related substrates with varying dependencies on GPVI and α2β1. To inform on Revacept binding to collagen, we used fluorescent-labelled anti-GPVI nanobody-28. In this first comparison of four inhibitors of platelet-collagen interactions with antithrombotic potential, we find that at arterial shear rate: (1) the thrombus-inhibiting effect of Revacept was restricted to highly GPVI-activating surfaces; (2) 9O12-Fab consistently but partly inhibited thrombus size on all surfaces; (3) effects of GPVI-directed interventions were surpassed by Syk inhibition; and (4) α2β1-directed intervention with 6F1 mAb was strongest for collagens where Revacept and 9O12-Fab were limitedly effective. Our data hence reveal a distinct pharmacological profile for GPVI-binding competition (Revacept), GPVI receptor blockage (9O12-Fab), GPVI signaling (PRT-060318), and α2β1 blockage (6F1 mAb) in flow-dependent thrombus formation, depending on the platelet-activating potential of the collagen substrate. This work thus points to additive antithrombotic action mechanisms of the investigated drugs.

Mice Lacking PECAM-1 and Ceacam1 Have Enhanced Platelet Secretion and Thrombus Growth: Novel Link with PAR4.

The Ig-ITIM bearing receptors, PECAM-1 and CEACAM1, have been shown net negative regulators of platelet-collagen interactions and hemiITAM signaling pathways. In this study, a double knockout (DKO) mouse was developed with deleted PECAM-1 and CEACAM1 to study their combined contribution in platelet activation by glycoprotein VI, C-type lectin-like receptor 2, protease activated receptor (PAR4), ADP purinergic receptors, and thromboxane receptor (TP) A2 pathways. In addition, their collective contribution was examined in thrombus formation under high shear and microvascular thrombosis using in vivo models. DKO platelets responded normally to ADP purinergic receptors and the TP A2 pathway. However, DKO platelets released significantly higher amounts of P-selectin compared with hyper-responsive Pecam-1-/- or Ceacam1-/- versus wild-type (WT) upon stimulation with collagen-related peptide or rhodocytin. In contrast, DKO platelets showed increased amounts of P-selectin exposure upon stimulation with PAR4 agonist peptide or thrombin but not Pecam-1-/- , Ceacam1-/- , or WT platelets. Blockade of phospholipase C (PLC) or Rho A kinase revealed that DKO platelets enhanced α-granule release via PAR4/Gαq/PLC signaling without crosstalk with Src/Syk or G12/13 signaling pathways. Severely delayed clot retraction in vitro was observed in DKO phenotype. The DKO model revealed a significant increase in thrombus formation compared with the hyper-responsive Ceacam1-/- or Pecam-1-/- versus WT phenotype. DKO platelets have similar glycoprotein surface expression compared with Pecam-1-/- , Ceacam1-/- , and WT platelets. This study demonstrates that PECAM-1 and CEACAM1 work in concert to negatively regulate hemiITAM signaling, platelet-collagen interactions, and PAR4 Gαq protein- coupled signaling pathways. Both PECAM-1 and CEACAM1 are required for negative regulation of platelet activation and microvascular thrombosis in vivo.

Molecular underpinnings of integrin binding to collagen-mimetic peptides containing vascular Ehlers–Danlos syndrome–associated substitutions

Collagens carry out critical extracellular matrix (ECM) functions by interacting with numerous cell receptors and ECM components. Single glycine substitutions in collagen III, which predominates in vascular walls, result in vascular Ehlers-Danlos syndrome (vEDS), leading to arterial, uterine, and intestinal rupture and an average life expectancy of <50 years. Collagen interactions with integrin α2β1 are vital for platelet adhesion and activation; however, how these interactions are impacted by vEDS-associated mutations and by specific amino acid substitutions is unclear. Here, we designed collagen-mimetic peptides (CMPs) with previously reported Gly → Xaa (Xaa = Ala, Arg, or Val) vEDS substitutions within a high-affinity integrin α2β1-binding motif, GROGER. We used these peptides to investigate, at atomic-level resolution, how these amino acid substitutions affect the collagen III-integrin α2β1 interaction. Using a multitiered approach combining biological adhesion assays, CD, NMR, and molecular dynamics (MD) simulations, we found that these substitutions differentially impede human mesenchymal stem cell spreading and integrin α2-inserted (α2I) domain binding to the CMPs and were associated with triple-helix destabilization. Although an Ala substitution locally destabilized hydrogen bonding and enhanced mobility, it did not significantly reduce the CMP-integrin interactions. MD simulations suggested that bulkier Gly → Xaa substitutions differentially disrupt the CMP-α2I interaction. The Gly → Arg substitution destabilized CMP-α2I side-chain interactions, and the Gly → Val change broke the essential Mg2+ coordination. The relationship between the loss of functional binding and the type of vEDS substitution provides a foundation for developing potential therapies for managing collagen disorders.

Deciphering the Roles of N-Glycans on Collagen-Platelet Interactions.

Collagen is a potent agonist for platelet activation, presenting itself as a key contributor to coagulation via interactions with platelet glycoproteins. The fine details dictating platelet-collagen interactions are poorly understood. In particular, glycosylation could be a key determinant in the platelet-collagen interaction. Here, we report an affinity purification coupled to a mass spectrometry-based approach to elucidate the function of N-glycans in dictating platelet-collagen interactions. By integrative proteomic and glycoproteomic analysis of collagen-platelet interactive proteins with N-glycan manipulation, we demonstrate that the interaction of platelet adhesive receptors with collagen is highly N-glycan regulated, with glycans on many receptors playing positive roles in collagen binding, with glycans on other platelet glycoproteins exhibiting inhibitory roles on the binding to collagen. Our results significantly enhance our understanding of the details of glycans influencing the platelet-collagen interaction.

Targeting Platelet GPVI Plus rt-PA Administration but Not α2β1-Mediated Collagen Binding Protects against Ischemic Brain Damage in Mice.

Platelet collagen interactions at sites of vascular injuries predominantly involve glycoprotein VI (GPVI) and the integrin α2β1. Both proteins are primarily expressed on platelets and megakaryocytes whereas GPVI expression is also shown on endothelial and integrin α2β1 expression on epithelial cells. We recently showed that depletion of GPVI improves stroke outcome without increasing the risk of cerebral hemorrhage. Genetic variants associated with higher platelet surface integrin α2 (ITGA2) receptor levels have frequently been found to correlate with an increased risk of ischemic stroke in patients. However until now, no preclinical stroke study has addressed whether platelet integrin α2β1 contributes to the pathophysiology of ischemia/reperfusion (I/R) injury. Focal cerebral ischemia was induced in C57BL/6 and Itga2−/− mice by a 60 min transient middle cerebral artery occlusion (tMCAO). Additionally, wild-type animals were pretreated with anti-GPVI antibody (JAQ1) or Fab fragments of a function blocking antibody against integrin α2β1 (LEN/B). In anti-GPVI treated animals, intravenous (IV) recombinant tissue plasminogen activator (rt-PA) treatment was applied immediately prior to reperfusion. Stroke outcome, including infarct size and neurological scoring was determined on day 1 after tMCAO. We demonstrate that targeting the integrin α2β1 (pharmacologic; genetic) did neither reduce stroke size nor improve functional outcome on day 1 after tMCAO. In contrast, depletion of platelet GPVI prior to stroke was safe and effective, even when combined with rt-PA treatment. Our results underscore that GPVI, but not ITGA2, is a promising and safe target in the setting of ischemic stroke.

Switching from abacavir to tenofovir disoproxil fumarate is associated with rises in soluble glycoprotein VI, suggesting changes in platelet-collagen interactions.

Altered platelet function has been proposed as an underlying mechanism to explain increased risk of myocardial infarction in people living with HIV associated with use of the nucleoside reverse transcriptase inhibitor abacavir (ABC). We aimed to examine changes in platelet biomarkers in people living with HIV switching from ABC. In a prospective, 48-week substudy of virally suppressed HIV-1-positive subjects randomized to remain on ABC/lamivudine (ABC/3TC) or switch to tenofovir disoproxil fumarate/emtricitabine, we measured soluble glycoprotein VI (sGPVI), soluble P-selectin, soluble CD40 ligand and von Willebrand factor in plasma collected over time and assessed differences using mixed effect models. Of 312 randomized participants, 310 were included in the analysis. Mean (SD) age 46.4 (9.3) years, 262 (85%) men and 201 (65%) white. At baseline, there was no significant between-group difference in sGPVI [tenofovir disoproxil fumarate/emtricitabine 3.75 (0.25) versus ABC/3TC 3.61 (0.22) ng/ml, P = 0.69]. Greater increases in sGPVI from baseline to week 48 occurred in those switched from ABC/3TC (effect size +0.57 ng/ml; 95% confidence interval, 0.2-0.94; P = 0.003). There was no significant baseline difference or change overtime in soluble P-selectin, soluble CD40 ligand or von Willebrand factor between groups. The significant increases in sGPVI that occur with a switch from ABC/3TC are suggestive of changes in platelet function centred on platelet/collagen interactions and potentially represent an underlying mechanism to explain increased risk of myocardial infarction with ABC.

Anti-thrombotic efficacy of S007-867: Pre-clinical evaluation in experimental models of thrombosis in vivo and in vitro

Pharmacological inhibition of platelet collagen interaction is a promising therapeutic strategy to treat intra-vascular thrombosis. S007-867 is a novel synthetic inhibitor of collagen-induced platelet aggregation. It has shown better antithrombotic protection than aspirin and clopidogrel with minimal bleeding tendency in mice. The present study is aimed to systematically investigate the antithrombotic efficacy of S007-867 in comparison to aspirin and clopidogrel in vivo and to delineate its mechanism of action in vitro. Aspirin, clopidogrel, and S007-867 significantly reduced thrombus weight in arterio-venous (AV) shunt model in rats. In mice, following ferric chloride induced thrombosis in either carotid or mesenteric artery; S007-867 significantly prolonged the vessel occlusion time (1.2-fold) and maintained a sustained blood flow velocity for >30 min. Comparatively, clopidogrel showed significant prolongation in TTO (1.3-fold) while aspirin remained ineffective. Both S007-867 and aspirin did not alter bleeding time in either kidney or spleen injury models, and thus maintained hemostasis, while clopidogrel showed significant increase in spleen bleeding time (1.7-fold). The coagulation parameters namely thrombin time, prothrombin time or activated partial thromboplastin time remained unaffected even at high concentration of S007-867 (300 µM), thus implying its antithrombotic effect to be primarily platelet mediated. S007-867 significantly inhibited collagen-mediated platelet adhesion and aggregation in mice ex-vivo. Moreover, when blood was perfused over a highly thrombogenic combination of collagen mimicking peptides like CRP-GFOGER-VWF-III, S007-867 significantly reduced total thrombus volume or ZV50 (53.4 ± 5.7%). Mechanistically, S007-867 (10–300 μM) inhibited collagen-induced ATP release, thromboxane A2 (TxA2) generation, intra-platelet [Ca+2] flux and global tyrosine phosphorylation including PLCγ2. Collectively the present study highlights that S007-867 is a novel synthetic inhibitor of collagen induced platelet activation, that effectively maintains blood flow velocity and delays vascular occlusion. It inhibits thrombogenesis without compromising hemostasis. Therefore, S007-867 may be further developed for the treatment of thrombotic disorders in clinical settings.

A Novel Microfluidic Assay to Determine Von Willebrand Factor Levels and Bleeding Phenotypes in Patients with Von Willebrand Disease and Mucocutaneous Bleeding

BackgroundVon Willebrand's disease (VWD) is the most common inherited bleeding disorder. Conventional assays cannot accurately detect all abnormalities in von Willebrand factor (VWF)-platelet-collagen interactions or predict a bleeding phenotype in patients with VWD.ObjectiveWe hypothesized that a microfluidic assay (MFA) could complement current assays and better correlate with bleeding.MethodsPatients: Patients with VWD or mucocutaneous bleeding (MCB) are enrolled in an IRB-approved study. Bleeding scores (BS) are obtained via the Vincenza bleeding assessment tool. In this analysis, Type 1 VWD is defined by a VWF Antigen (VWF:Ag) level <40 IU/dL and a VWF activity (VWF:RCo)/VWF:Ag ratio >0.6, Type II VWD has a VWF:RCo/VWF:Ag ratio of <0.6, and Type III disease has VWF:Ag and VWF:RCo levels <10 IU/dL. MCB is defined as clinical mucocutaneous bleeding, a VWF:Ag level >40 IU/dL and no other laboratory evidence of a diagnosable bleeding diathesis.Standard Assays: Samples are assayed for platelet count, platelet aggregometry (agonists: collagen/ristocetin/ADP), VWF:Ag, VWF:RCo, Type III collagen binding (VWF:CBIII) and PFA-100 closure time (ADP and EPI cartridges).MFA: C ollagen (500 µg/mL) is patterned onto a glass substrate and whole blood is perfused at three distinct shear rates, low (150s-1), mid (750s-1), and high (1500s-1) for five minutes. Images of platelet adhesion are collected every 3 seconds at pre-defined points.Data Processing: Total platelet surface coverage is determined and graphed over time. Two outputs are evaluated, (1) the lag time: the time to reach 5% surface coverage and (2) the slope: slope during the linear portion of platelet adhesion.ResultsPatients: 42 samples were obtained from 35 patients with VWD/MCB. 11/35 (31%) are from type 1 VWD patients, 2/35 (6%) are from type 2 VWD patients, 1/35 (3%) are from a Type 3 VWD patients, and 22/35 (60%) are from patients with MCB.Assay Correlation: In all patients, the midshear slope correlated better with VWF:Ag (R2 = 0.52 P<0.0001) than the PFA100-ADP (R2 = 0.28 P=0.0008) and PFA100-EPI (R2 =0.32 P=0.0002) (Table 1). The midshear slope (R2 = 0.52 P=< 0.0001) also outperformed the PFA100-ADP (R2 = 0.33 P= 0.0003) and PFA100-EPI (R2 = 0.39 P= < 0.0001) in correlating with VWF:RCo levels (Table 1). In the Type 1 VWD cohort, the mid shear slope correlated with VWF:Ag (R2 = 0.54 P=0.01) (figure 1). In comparison, the PFA-100 ADP/EPI was relatively insensitive to VWF:Ag levels <40 IU/dL (figure 1). These results show that a single MFA assay correlates with VWF:Ag and VWF:RCo levels better than the PFA-100 closure time in a cohort of patients with VWD/MCB and is more sensitive to VWF:Ag levels <40 IU/dL in a cohort of individuals with Type 1 VWD.BS Correlation: No clinical assays or MFA output significantly correlated with BS in any cohort analyzed.Response to therapy: In 4 patients pre and 1hr post DDAVP, the MFA lag time remained the same or decreased (low=15.75s to 15.75s, mid=18s to 11.25s, high=30.75s to 9s) and the slope increased (low=0.0044 to 0.0057, mid=0.0044 to 0.0066, high=0.0037 to 0.0059). The increased low shear slope, 0.0044 to 0.0057, was statistically significant (p= 0.0244). This suggests that a MFA may be useful to determine response to DDAVP therapy.ConclusionsA novel collagen-based MFA outperformed the PFA100 ADP/EPI in correlating with VWF:Ag and VWF:RCo in patients with VWD/MCB. In type 1 VWD patients, it also demonstrated a significant correlation with VWF:Ag levels and a more linear relationship than the PFA100 ADP/EPI. Finally, the MFA was able to determine a response to DDAVP therapy. Thus a MFA, with a small sample input (150 µL) as compared to other shear dependent assays such as the PFA100 (1000 µL), may be useful to determine multiple clinically relevant parameters in situations where blood samples are difficult to obtain such as in neonatal settings or poor venous access.Table 1Comparison of R2 values Between Standard Assays and Shear-based Assays in All Patients with VWD/MCB.Standard AssayShear-Based AssayR2 ValueP ValueVWF:AgPFA100 (Collagen/EPI)0.320.0002PFA100 (Collagen/ADP)0.280.0008MFA Mid-shear Slope0.52<0.0001VWF:RCoPFA100 (Collagen/EPI)0.39< 0.0001PFA100 (Collagen/ADP)0.330.0003MFA Mid-shear Slope0.52< 0.0001 [Display omitted] DisclosuresNg:CSL Behring: Consultancy; Baxter: Consultancy. Manco-Johnson:Baxter, bayer, biogen, CSL Behring, NovoNordish: Honoraria.

Platelet mechanosensing of collagen matrices.

During vascular injury, platelets adhere to exposed subendothelial proteins, such as collagen, on the blood vessel walls to trigger clot formation. Although the biochemical signalings of platelet-collagen interactions have been well characterized, little is known about the role microenvironmental biomechanical properties, such as vascular wall stiffness, may have on clot formation. To that end, we investigated how substrates of varying stiffness conjugated with the same concentration of Type I collagen affect platelet adhesion, spreading, and activation. Using collagen-conjugated polyacrylamide (PA) gels of different stiffnesses, we observed that platelets do in fact mechanotransduce the stiffness cues of collagen substrates, manifesting in increased platelet spreading on stiffer substrates. In addition, increasing substrate stiffness also increases phosphatidylserine exposure, a key aspect of platelet activation that initiates coagulation on the platelet surface. Mechanistically, these collagen substrate stiffness effects are mediated by extracellular calcium levels and actomyosin pathways driven by myosin light chain kinase but not Rho-associated protein kinase. Overall, our results improve our understanding of how the mechanics of different tissues and stroma affect clot formation, what role the increased vessel wall stiffness in atherosclerosis may directly have on thrombosis leading to heart attacks and strokes, and how age-related increased vessel wall stiffness affects hemostasis and thrombosis.

Platelets and Arterial Thrombosis: Evolving Role of Platelet Gpvi as an Antithrombotic Drug Target

Acute coronary syndrome is one of the leading causes of mortality and morbidity worldwide. Although the recent advancements in existing anti-platelet therapeutics have significantly reduced the burden of cardiovascular deaths but owing to their global impact on primary haemostasis, their clinical benefits are often limited by bleeding risk. Recent developments in the understanding of platelet biology have placed platelet-collagen interaction at the critical position in the sequence of events during arterial thrombosis. Thus there is considerable interest in developing novel therapeutic strategies against platelet collagen receptor glycoprotein VI (GPVI) because not only its selective blockade or deficiency holds promise for the reduced risk of bleeding complications as evidenced in mice and humans, but also the exclusive expression of GPVI on platelets and megakaryocytes offers to prevent the adverse off-target effects of antithrombotic drugs. The present review summarizes recent developments in understanding of platelet functions during arterial thrombosis and structural and functional attributes of platelet GPVI to identify it as a novel antithrombotic drug target. Moreover, it also emphasises on the development of GPVI antagonists using various experimental approaches as well as their prospective use in cardiovascular disorders.

Antiplatelet Effects of Caffeic Acid Due to Ca2＋ MobilizationInhibition Via cAMP-Dependent Inositol-1, 4, 5-Trisphosphate Receptor Phosphorylation

In this study, we investigated the effects of caffeic acid (CAFA), a phenolic acid, on Ca(2＋)-antagonistic cyclic nucleotides associated with the phosphorylation of inositol 1,4,5-trisphosphate receptor (IP3R) and vasodilator-stimulated phosphoprotein (VASP) and the thromboxane A2 (TXA2)-associated microsomal cyclooxygenase-1 (COX-1) activity in collagen (10 μg/mL)-stimulated platelet aggregation. Washed platelets (10(8)/mL) obtained from Sprague-Dawley rats (6-7 weeks old, male) were preincubated for 3 minutes at 37℃ in the presence of 2 mM exogenous CaCl2 with or without CAFA or other materials, stimulated with collagen (10 μg/mL) for 5 minutes, then used to determine the levels of intracellular cytosolic Ca(2＋) ([Ca(2＋)]i), TXA2, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), COX-1 activity, VASP and IP3R phosphorylation. CAFA dose-dependently inhibited collagen-induced platelet aggregation and suppressed the production of TXA2, an aggregation-inducing autacoid associated with the strong inhibition of COX-1 in platelet microsomes exhibiting cytochrome C reductase activity. CAFA dose-dependently inhibited collagen-elevated [Ca(2＋)]i mobilization, which was increased by a cAMP-dependent protein kinase (A-kinase) inhibitor, Rp-8-Br-cAMPS, but not a cGMP-dependent protein kinase (G-kinase) inhibitor, Rp-8-Br-cGMPS. In addition, CAFA significantly increased the formation of cAMP and cGMP, intracellular Ca(2＋)-antagonists that function as aggregation-inhibiting molecules. CAFA increased IP3R (320 kDa) phosphorylation, indicating the inhibition of IP3-mediated Ca(2＋) release from internal stores (i.e. the dense tubular system) via the IP3R on collagen-activated platelets. Furthermore, CAFA-induced IP3R phosphorylation was strongly inhibited by an A-kinase inhibitor, Rp-8-Br-cAMPS, but only mildly inhibited by a G-kinase inhibitor, Rp-8-Br-cGMPS. These results suggest that the inhibition of [Ca(2＋)]i mobilization by CAFA is resulted from the cAMP/A-kinase-dependent phosphorylation of IP3R. CAFA elevated the phosphorylation of VASP-Ser(157), an A-kinase substrate, but not the phosphorylation of VASP-Ser(239), a G-kinase substrate. We demonstrate that CAFA increases cAMP and subsequently phosphorylates both IP3R and VASP-Ser(157) through A-kinase activation to inhibit [Ca(2＋)]i mobilization and TXA2 production via the inhibition of the COX-1 activity. These results strongly indicate that CAFA is a potent beneficial compound that elevates the level of cAMP-dependent protein phosphorylation in collagen-platelet interactions, which may result in the prevention of platelet aggregation-mediated thrombotic diseases.

Aegyptin inhibits collagen-induced coagulation activation in vitro and thromboembolism in vivo

Aegyptin is a mosquito salivary gland protein and potent inhibitor of platelet aggregation. Aegyptin binds to the von Willebrand factor-binding site on collagen and prevents its interaction with platelets. Because collagen also induces plasma clotting by activation of factor XII, we evaluated the effects of aegyptin on collagen-induced coagulation activation and how it interferes with thrombosis in three different in vivo models. Our results demonstrate that aegyptin abolishes collagen-induced clot formation and thrombin generation in platelet-free plasma. Aegyptin has no antithrombotic activity in the arteriovenous shunt model (collagen-independent) but it prevents laser-induced collagen-mediated thrombus formation in rats. Furthermore, aegyptin protects mice from collagen and epinephrine-induced thromboembolism. Therefore, aegyptin has a dual antithrombotic mechanism: inhibition of platelet-collagen interaction and collagen’s pro-coagulant activity. This is the first description of a collagen-binding protein that also inhibits collagen-mediated coagulant activity.

Measurement of the Interaction Between Recombinant I-domain from Integrin alpha 2 beta 1 and a Triple Helical Collagen Peptide with the GFOGER Binding Motif Using Molecular Force Spectroscopy

The role of the collagen-platelet interaction is of crucial importance to the haemostatic response during both injury and pathogenesis of the blood vessel wall. Of particular interest is the high affinity interaction of the platelet transmembrane receptor, alpha 2 beta 1, responsible for firm attachment of platelets to collagen at and around injury sites. We employ single molecule force spectroscopy (SMFS) using the atomic force microscope (AFM) to study the interaction of the I-domain from integrin alpha 2 beta 1 with a synthetic collagen related triple-helical peptide containing the high-affinity integrin-binding GFOGER motif, and a control peptide lacking this sequence, referred to as GPP. By utilising synthetic peptides in this manner we are able to study at the molecular level subtleties that would otherwise be lost when considering cell-to-collagen matrix interactions using ensemble techniques. We demonstrate for the first time the complexity of this interaction as illustrated by the complex multi-peaked force spectra and confirm specificity using control blocking experiments. In addition we observe specific interaction of the GPP peptide sequence with the I-domain. We propose a model to explain these observations.

Vimentin Exposed On Platelets Serves As an Adhesive Receptor for Von Willebrand Factor

Abstract 259Platelet adhesion, activation, and aggregation in the vasculature are necessary events in both life-saving hemostasis and pathological thrombosis. Thrombosis may occur in patients presenting with several clinical conditions including atherosclerosis, cardiovascular disease, and inflammation. Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that plays a critical role in mediating platelet adhesion, activation, and aggregation on the exposed subendothelium in order to maintain hemostasis under arterial flow conditions. On the other hand, VWF permits the stabilization of platelets adherent to components of ruptured atherosclerotic plaques, leading to artery-occluding thrombus formation. The initial interaction of activated or hyperadhesive VWF with platelets occurs via the interaction between the A1 domain of VWF and the platelet receptor glycoprotein (GP)Ibα. This engagement is responsible for reducing the velocity of rapidly flowing platelets, allowing the rolling platelets to interact with the second binding site on VWF for the platelet receptor GPIIb/IIIa; a binding site that is located within the C domains of VWF. Therefore, the hyperadhesive property of VWF apparently relies on the synchronized interaction of the two platelet surface receptors, GPIbα and GPIIb/IIIa. Despite this concept, we and others have speculated that other binding site in VWF synergistically works with the A1 domain to quickly capture the extremely fast flowing platelets. We have obtained interesting results from studies using a monomeric A1A2A3 domain protein that lacks the binding site for GPIIb/IIIa. For example, the rolling velocity of platelets over an A1A2A3-coated surface was markedly lower than that seen with use of the single A1 domain. This observation suggests the possibility of an additional binding site in the A domains for platelets. Given the similar hyperadhesive features of the A1A2A3 protein and plasma VWF, we proposed to look for a potential receptor on platelets with a recognition site within the A domains of VWF. We suggested examining vimentin because, it was identified as a binding protein for the isolated A2 domain of VWF in our laboratory, and vimentin has been found on the surface of platelets. First, both full length VWF and recombinant A1A2A3 proteins efficiently bound to human vimentin only in the presence of the modulator ristocetin, indicating that vimentin preferably interact with the active conformation of VWF. In fact, a constitutively active A1A2A3 protein (containing a gain-of-function mutation in A1 domain) had a binding activity for vimentin higher than that of wild type (WT) A1A2A3 in the absence of ristocetin. Second, anti-vimentin monoclonal antibody blocked the interaction of that mutant A1A2A3 to activated washed platelets using flow cytometry. Third, we then examined the effect of anti-vimentin antibody on flow-dependent platelet adhesion to A1A2A3-coated surface at high shear stress. In comparison to whole blood incubated with irrelevant IgG molecule as a negative control, the anti-vimentin antibody blocked 75% platelet adhesion to the triple-A domain protein. Finally, whole blood from vimentin-deficient or WT mice was perfused over a surface coated with murine VWF at high shear rate. In comparison to platelets from WT mice, vimentin-deficient platelets had a significant reduced platelet adhesion to VWF (25% of WT). Similarly, vimentin-deficient platelets had a reduced platelet adhesion to collagen (20% of WT) under high flow conditions. This platelet-collagen interaction is initially mediated by VWF. These interesting results indicate that vimentin on platelets serves as a receptor for VWF, and this binding may participate in the initial interaction of circulating platelets with VWF under flow conditions. Disclosures:No relevant conflicts of interest to declare.

Evaluation of Primary Haemostasis in Patients Undergoing Cardiac Surgery Using a Novel Automated Microchip Flow-Chamber System (T-TAS).

AbstractAbstract 2171Post operative bleeding is a common complication of cardiac surgery; its aetiology may be multifactorial but is often attributed to platelet dysfunction. However, at present no reliable point of care test (POCT) is available for this and patients are often managed empirically; approximately 40% receive pooled platelet transfusions but many require re-sternotomy. An automated microchip flow chamber system that directly measures platelet-collagen interaction under shear (1500s−1) has recently been developed (T-TAS; Total Thrombus-formation system; Zakros, Fujmori Kogyto, Japan). We hypothesized that this could be developed as a POCT to assess platelet function under shear, allowing targeted and more rational use of blood products. We used this system to study 20 patients aged 19–72 years, before and after cardiac surgery. The T-TAS was used to determine the onset of occlusion (time to 10kPa, T10) and primary haemostatic potential (AUCend-area under pressure curve). Haematocrit (Hct), platelet count and von Willebrand factor (VWF) levels and thromboelastography (TEG) were measured at the corresponding time points.Surgery resulted in a significant increase in time to occlusion onset (T10) (p=0.006; n=20, Figure 1A). Similarly, the AUCend significantly decreased after surgery (p=0.001, n=19; Figure 1B) with the majority of cases (12 out of 20) failing to occlude within 30 minutes. The change in T10 inversely correlated with change in AUCend(r=-0.5, p=0.02).VWF is essential for platelet adhesion to collagen under high shear stress. Higher VWF levels appeared to be associated with lower T10, but this was not significant and only observed prior to surgery. Moreover, no significant correlation between the change in T10 or AUCend and changes in VWF pre- and post-surgery was observed.Surprisingly, pre and post-operative hematocrit were both negatively correlated with AUCend (r=-0.5, p=0.03, n=11 and r=-0.5, p=0.02,n=11 respectively).No correlation was observed between platelet count and the T-TAS parameters. However, the change in AUCend (but not T10) was shown to correlate with the change in platelet count before and after surgery (r = 0.58, p = 0.01).Finally, T10 and AUCend values were not significantly altered in patients given anti-platelet drugs: aspirin and/or clopidrogel. This implies that the T-TAS is sensitive to the initial adhesion of platelets to VWF-collagen rather than to platelet activation and aggregation.These data demonstrate that cardiac bypass surgery affects VWF mediated platelet adhesion to collagen under flow and this can be detected using a microchip flow chamber system. The lack of correlation with VWF levels and apparently paradoxical effect of haematocrit suggest that the dominant mechanism for the impaired postoperative haemostasis is a decline in platelet function and in particular a decrease in adhesive function. This is likely to result from altered exposure and/or function of GPIb resulting from surgery and cardio-pulmonary bypass.Current POCTs are largely insensitive to primary haemostatic mechanisms, while conventional laboratory measures of primary haemostasis are non-physiological, and require a considerable expertise and time. As expected, TEG gave similar values pre and post-surgery for all subjects, indicating that a flow system is required to adequately evaluate primary haemostasis. The advantages of this novel automated microchip flow chamber system are the small blood volume required and the rapidity of results. Our data suggest a place for this system in a point-of care setting, providing useful assessment of haemostatic status following surgery and with the potential to improve patient management. Further studies are warranted. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Abstract 494: Platelet Aggregation in a Model of Simulated Arterial Flow: Red Blood Cells Are Integral Players in Hemostatic Control Resuscitation

Background: Approximately one in four trauma patients exhibits an early bleeding diathesis, termed the acute coagulopathy of trauma and defined by an initial INR &gt;1.2, which is associated with a 3-fold increase in mortality. In recent years, the concept of damage control resuscitation has been developed to address this problem. This approach emphasizes hemorrhage control and early resuscitation with blood products in a 1:1:1 ratio of plasma:platelets:RBCs in an attempt to replace whole blood lost in hemorrhage. The optimal ratio of blood components has not been established and few pre-clinical studies have explored this problem under arterial shear conditions. Objective: Our objective is to establish stepped ranges of hematocrit values and platelet counts to identify the component and concentration of that component most responsible for the largest loss of platelet-collagen interaction. Methods: Donor citrated whole blood containing calcein tagged platelets was flowed at an arterial shear rate of 1000/second through a 70μm x 350μm x 5mm collagen-coated chamber at 37°C for 10 min, n=6. Images were captured every 15 seconds and analyzed for fluorescence intensity. Results: Linear regression analysis generated slopes (fluorescent intensity units/second) that were most different as hematocrit dropped below physiologic levels (HCT40control=100%, HCT25=39.5%, HCT20=21.4%, HCT18=17.4%, HCT16=11.6%). Non-linear fitting of slopes vs. HCT yielded the equation y=1.27e4 x 2 -3.51e4 x +4.43e5 describing an exponential decrease in platelet-collagen interaction with linear decreases in HCT. Conclusions: The largest drop in platelet adherence to collagen in a physiological model of arterial flow occurs as hematocrit falls from 40%. The commonly suggested 30HCT target for minimal RBC transfusion during resuscitation may not adequately support hemostasis and should be reconsidered.

Diesel exhaust particles impair platelet response to collagen and are associated with GPIbα shedding

ObjectiveAir pollution with fine particulates (PM10 and PM2.5) is associated with an increased incidence of cardiovascular events. The proposed mechanisms include indirect proinflammatory and procoagulant reactions involving activation of pulmonary macrophages, endothelial cells and the TNF/TF pathway, or direct procoagulant effects. Our laboratory has observed a reduction of the platelet responsiveness to collagen after exposure to diesel exhaust particles (DEP). HypothesisDEP directly interfere with platelet–collagen interactions by selectively inducing the shedding of platelet signaling receptors via metalloproteinases, which would represent a novel mechanism for DEP action on platelets. MethodsCitrated blood from healthy volunteers was exposed to highly standardized DEP at concentrations of 0.1, 2.5 and 5.0μg/ml or ultrafine carbon black (ufCB, 0.1μg/ml) and the plasmatic and platelet response was analysed. The closure times with the PFA-100 device and the platelet aggregation in response to a variety of agonists were monitored. Interleukins (IL)-1β and IL-8 levels were determined by ELISA and soluble P-selectin by the Luminex bead assay. Thrombin activity was measured as the endogenous thrombin potential (ETP) by fluorescence spectrometry. Soluble GPVI and GPIbα (glycocalicin) ectodomain fragments were measured by ELISA. ADAMTS13 activity was determined by a FRETS based assay and plasmin activity with Spectrozyme PL. ResultsAggregation assays where platelets were treated with low dose DEP or ultrafine carbon black (ufCB) revealed a significantly increased response to low doses of collagen (p<0.05, n=5). At higher doses, however, collagen induced aggregation was suppressed by DEP treatment: at 2.5μg/ml, the inhibition was 34±12% (p<0.01, n=10). Aggregations with cross-linked collagen related peptide (CRPxl), convulxin and with the monoclonal antibody 9O12.2 (all known to specifically bind to and activate GPVI) were also diminished. Ristocetin, arachidonic acid and ADP responses were normal at all DEP concentrations. No cleavage of GPVI ectodomain was detected (soluble GPVI 27.8±3 vs. 28±4μg/ml mean±SEM, n=10); however increased plasma glycocalicin (GPIbα ectodomain) was detected upon diesel exposure (2.58±0.11 vs. 2.28±0.03μg/ml p<0.01, n=10). ADAMTS13 and plasmin activity remained unaffected by DEP under the conditions tested. Platelets were not activated by either DEP or ufCB as soluble P-selectin was insensitive to these. ConclusionsDEP specifically and directly interferes with platelet–collagen interactions. The functional consequences are biphasic and include enhance platelet aggregation at lower DEP concentrations and inhibition at a higher dose. Our data indicate that this interaction does not involve P-selectin or GPVI shedding. It is however associated with an increase in GPIb cleavage.

Epigallocatechin-3-Gallate Has an Anti-Platelet Effect in a Cyclic AMP-Dependent Manner

In this study, we investigated the effect of (-)-epigallocatechin-3-gallate (EGCG) on cyclic nucleotide production and vasodilator-stimulated phosphoprotein (VASP) phosphorylation in collagen (10 µg/mL)-stimulated platelet aggregation. Washed platelets (10(8)/mL) from Sprague-Dawley rats (6-7 weeks old, male) were preincubated for 3 min at 37°C in the presence of 2 mM exogenous CaCl(2) with or without EGCG or other materials, stimulated with collagen (10 µg/mL) for 5 min, and then used for the determination of intracellular cytosolic Ca(2+) ([Ca(2+)](i)), thromboxane A(2) (TXA(2)), adenosine 3',5'-cyclic monophosphate (cAMP), guanosine 3',5'-cyclic monophosphate (cGMP), and VASP phosphorylation. EGCG dose-dependently inhibited collagen-induced platelet aggregation by inhibiting both [Ca(2+)](i) mobilization and TXA(2) production. Of two aggregation-inhibiting molecules, cAMP and cGMP, EGCG significantly increased intracellular levels of cAMP, but not cGMP. EGCG-elevated cAMP level was decreased by SQ22536, an adenylate cyclase inhibitor, but not by etazolate, a cAMPspecific phosphodiesterase inhibitor. In addition, EGCG elevated the phosphorylation of VASP-Ser(157), a cAMP-dependent protein kinase (A-kinase) substrate, but not the phosphorylation of VASP-Ser(239), a cGMP-dependent protein kinase substrate, in intact platelets and collagen-induced platelets, and VASP-Ser(157) phosphorylation by EGCG was inhibited by both an adenylate cyclase inhibitor SQ22536 and an A-kinase inhibitor Rp-8-Br-cAMPS. We have demonstrated that EGCG increases cAMP via adenylate cyclase activation and subsequently phosphorylates VASP-Ser(157) through A-kinase activation to inhibit [Ca(2+)](i) mobilization and TXA(2) production on collagen-induced platelet aggregation. These results strongly indicate that EGCG is a beneficial compound elevating cAMP level in collagen-platelet interaction, which may result in the prevention of platelet aggregation-mediated thrombotic diseases.