Shear-induced Platelet Adhesion Research Articles

Microfluidic assay of antiplatelet agents for inhibition of shear-induced platelet adhesion and activation.

We have developed a microfluidic system to perfuse whole blood through a flow channel with an upstream stenotic region and a downstream protein capture region. This flow-based system was used to assay how effectively antiplatelet agents suppress shear-induced platelet adhesion and activation downstream of the stenotic region. Microcontact printing was used to covalently attach one of three platelet binding proteins [fibrinogen, collagen, or von Willebrand factor (vWf)] to the surface of the downstream capture region. Whole blood with an antiplatelet agent was transiently exposed to an upstream high wall shear rate (either 4860 s-1 or 11 560 s-1), and subsequently flowed over the downstream capture region where the platelet adhesion was measured. Several antiplatelet agents (acetylsalicylic acid, tirofiban, eptifibatide, anti-vWf, and anti-GPIbα) were evaluated for their efficacy in attenuating downstream adhesion. Following antibody blocking of vWf or GPIbα, downstream platelet activation was also assessed in perfused blood by flow cytometry using two activation markers (active GPIIb/IIIa and P-selectin). Acetylsalicylic acid demonstrated its inability to diminish shear-induced platelet adhesion to all three binding proteins. GPIIb/IIIa inhibitors (tirofiban and eptifibatide) significantly reduced platelet adhesion to fibrinogen. Antibody blocking of vWf or GPIbα effectively diminished platelet adhesion to all three capture proteins as well as platelet activation in perfused blood, indicating an essential role of vWf-GPIbα interaction in mediating shear-induced platelet aggregation.

Device-Induced Hemostatic Disorders in Mechanically Assisted Circulation

Mechanically assisted circulation (MAC) sustains the blood circulation in the body of a patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) or on ventricular assistance with a ventricular assist device (VAD) or on extracorporeal membrane oxygenation (ECMO) with a pump-oxygenator system. While MAC provides short-term (days to weeks) support and long-term (months to years) for the heart and/or lungs, the blood is inevitably exposed to non-physiological shear stress (NPSS) due to mechanical pumping action and in contact with artificial surfaces. NPSS is well known to cause blood damage and functional alterations of blood cells. In this review, we discussed shear-induced platelet adhesion, platelet aggregation, platelet receptor shedding, and platelet apoptosis, shear-induced acquired von Willebrand syndrome (AVWS), shear-induced hemolysis and microparticle formation during MAC. These alterations are associated with perioperative bleeding and thrombotic events, morbidity and mortality, and quality of life in MCS patients. Understanding the mechanism of shear-induce hemostatic disorders will help us develop low-shear-stress devices and select more effective treatments for better clinical outcomes.

Chronic Lymphocytic Leukaemia Is Associated with a Broad Platelet-Function Defect Which Is Exacerbated By Ibrutinib and Acalabrutinib

INTRODUCTIONBleeding from thrombocytopathy is a common complication of advanced chronic lymphocytic leukaemia (CLL). In addition to disease-related thrombocytopenia, the presence of the CLL clone and/or therapeutic interventions may further impair platelet function. In particular, the BTK inhibitors ibrutinib and acalabrutinib are known to inhibit platelet glycoprotein VI (GPVI)-mediated platelet aggregation. We compared platelet function and markers of GPVI activation between untreated CLL patients, ibrutinib-treated CLL patients and healthy controls, and studied the in vitro effects of ibrutinib and acalabrutinib on clinically utilised platelet function assays to assess their impact on GPVI-mediated as well as non-GPVI-mediated platelet activation pathways.METHODSBlood samples from 17 healthy volunteers and 8 untreated CLL patients were spiked with vehicle or comparable plasma concentrations of ibrutinib (0.3µM, 1.0µM) and acalabrutinib (1.8µM, 6.0µM) attainable during the treatment of CLL. Additional samples were obtained from 5 CLL patients undergoing ibrutinib treatment. Platelet function was evaluated using whole blood multiple electrode aggregometry (MEA - Multiplate®) and light transmission aggregometry (LTA - AggRAM®) in response to varying concentrations of aggregation-inducing reagents (collagen, CRP-XL, ADP, TRAP, ristocetin, arachidonic acid, and adrenaline). Shear-induced platelet adhesion was assessed using PFA-100®. Soluble GPVI plasma levels were assessed by ELISA.RESULTSIn the absence of treatment, CLL patients exhibited significant platelet defects on whole-blood platelet function analyses in response to various agonists including ADP, ristocetin, TRAP and collagen (MEA) and prolongation of PFA-100® collagen/epinephrine closure time. This impairment was not replicated in assays using platelet-rich plasma (LTA). Ibrutinib-treated CLL patients demonstrated an additive impairment of platelet function, especially in regards to collagen-mediated activation by MEA or PFA-100®. There was no significant difference in soluble GPVI levels between normal, untreated or ibrutinib treated CLL patients.Addition of clinically-attainable concentrations of ibrutinib and acalabrutinib in vitro produced similar concentration-dependent inhibition of platelet function in healthy controls, with inhibition of aggregation evident in response to various agonists including collagen, CRP-XL, ristocetin and ADP but not arachidonic acid or TRAP. Ibrutinib also impaired aggregation in response to epinephrine, and caused selective prolongation of the PFA-100® collagen/epinephrine closure time, an effect not observed with acalabrutinib. MEA appears more sensitive and reproducible than LTA to describe the various inhibitory effects on platelet aggregation. Similar concentration-dependent inhibition of platelet function was observed by adding ibrutinib and acalabrutinib in vitro to blood samples from untreated CLL patients.CONCLUSIONSCLL is associated with a broad platelet function defect, which can be exacerbated by BTK inhibitors. Acalabrutinib induces a platelet function defect similar but less potent to that observed with ibrutinib, with the exception of shear-induced platelet adhesion (PFA-100®) which was only abnormal with ibrutinib. Routine platelet function assays are capable of quantifying BTK inhibitor-induced platelet dysfunction in CLL patients, with the most sensitive and reproducible measure being collagen-induced aggregation by MEA. There was no evidence for BTK-dependent platelet GPVI cleavage. Whole-blood platelet function assays may have utility in managing CLL patients presenting with bleeding or requiring urgent surgery during therapy with BTK inhibitors. DisclosuresMcGregor:Pfizer: Other: Conference travel support; Bristol-Myers Squibb: Other: Conference travel support; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria. Baker:CSL Behring: Research Funding; Biogen Idec: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Research Funding; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Research Funding; Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Other: Conference travel support, Research Funding; Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Conference travel support; Roche: Other: Conference travel support; Novo Nordisk: Other: Conference travel support.

Platelet-Delivered Recombinant ADAMTS13 Inhibits Platelet Adhesion and Aggregation on Collagen Surface Under Flow in the Presence of Autoantibodies from Acquired Thrombotic Thrombocytopenic Purpura

Background: Acquired thrombotic thrombocytopenic purpura (TTP) is a rare but potentially fatal syndrome. It is primarily caused by severe deficiency of plasma ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) activity. ADAMTS13 is a plasma metalloprotease that cleaves von Willebrand factor (VWF), thereby reducing the platelet adhesion and aggregation under flow. Plasma exchange is the only effective initial therapy for acquired autoimmune TTP. However, in hospital mortality rate remains as high as 10-20% in addition to the concern about the complications associated with the placement of central catheter and the use of plasma products. Therefore, a better and more effective therapy is urgently needed.Objectives: To develop a more effective therapy, we determined the uptake and package of recombinant ADAMTS13 (rA13) in platelets and assessed the efficacy of platelets-delivered rA13 in inhibiting platelet adhesion and aggregation and thrombus formation under arterial shear.Methods: Human platelets were isolated from whole blood, washed, and incubated with various concentrations of rA13 (1-100 microgram/ml) at 25 and 37 degree Celsius for various times. The amount of rA13 uptake by platelets was determined in cell lysate by Western blotting and FRETS-VWF73 assays and in fixed cells by immunofluoresent microscopy and flow cytometry. The function of platelet delivered rA13 was also determined by shear-induced platelet adhesion and aggregation on collagen surface using a microfluidic system.Results: Freshly isolated and blood bank stored platelets were able to update rA13 in a temperature, concentration, and time-dependent manner. When rA13 (5 microgram/ml) was incubated with platelets, nearly all platelets were positive for rA13, assessed by immunofluoresent microscopy and flow cytometry. The rA13 inside platelets remained intact and was proteolytically active in cleaving FRETS-VWF73. Confocal imaging revealed that rA13 was partially co-localized with VWF in alpha granules of platelets. Microfluidic assay demonstrated that platelet-delivered rA13 was able to dramatically inhibit platelet adhesion and aggregation on collagen-coated surface under arterial shear (100 dyne/cm2) in the absence and presence of a human monoclonal antibody against ADAMTS13 (scFv4-20) that was isolated from a patient with acquired autoimmune TTP. These results were consistent with the inhibitory effects observed with mouse transgenic platelets expressing rA13 under the same conditions. When 1/3 of whole blood Adamts13-/- platelets were replaced with transgenic platelets (containing rA13), the coverage of all fluorescent platelets onto a VWF-collagen surface was dramatically reduced (data not shown).Conclusion: Our results demonstrate that platelets uptake and deliver rA13 to the site of thrombus formation under flow and the platelet-delivered rA13 may be efficacious for treating acquired TTP with inhibitors. DisclosuresZheng:Ablynx: Consultancy; Alexion: Research Funding.

Interaction of Red Cell Microparticles (RMP) with Platelets: Potential Role of RMP in Hemostasis and Thrombosis,

Abstract 3263 Introduction:We previously reported data indicating that RMP are well suited for use as hemostatic agent for treating bleeding disorders (Jy et al, Hemophilia 17:4, 2011). Previous studies have shown that RMP can contribute to RBC-related thrombotic complications such as sickle cell disease and PNH. Microparticles (MP) derived from platelets (PMP), endothelia (EMP), and leukocytes (LMP) are believed to play a role in hemostasis and thrombosis. They can adhere to blood cells and endothelia, facilitating prothrombotic and proinflammatory reactions. However, less is known about interaction of RMP with cells and their potential role in hemostasis and thrombosis. Here we report evidence of interaction of RMP with platelets resulting in enhanced platelet aggregation and increased size of adherent platelet aggregates induced by shear stress. Methods:(i) RMP were prepared by high-pressure extrusion of washed RBC. (ii) Platelet aggregation was performed in a Chrono-log aggregometer. PRP (490 μL) was mixed with 10 μL of RMP (1 × 108 /mL final conc.) for 5 min, then low-dose activating agent (ADP 0.2 μM, or arachidonic acid (AA) 0.3 mM) was added. (iii) Shear-induced platelet adhesion was measured in a cone-and-well device (Diamed Impact-R). Whole blood was pre-incubated with RMP as above for 10 min, then subjected to various shear rates (900, 1800, 2700 sec−1) for 1 min. The adherent platelets were then washed, stained, and quantitated by image analyzer. (iv) RMP-platelet interaction employed 2-color flow cytometry. RMP-platelet conjugates were identified by co-expression of α-CD41-FITC and α-glycophrin A-PE, in both the free platelet and micro-aggregated platelet populations. Results:(1) Platelet aggregation: Addition of RMP to PRP did not induce platelet aggregation. However, RMP enhanced platelet aggregation induced by low-dose ADP or AA. Low-dose ADP alone induced a transient increase of aggregation peaking at 25–35% followed by slow disaggregation to 0–5% at 10 min; but in presence of RMP, a similar rate (slope) of aggregation was seen but peaking at 50–60% and disaggregation was abolished. Using AA, the RMP also potentiated aggregation from 20–30% to 50–60%. These results were obtained with heparinized PRP. Interestingly, when citrated PRP was used, the RMP effect was negligible. (2) Shear-induced platelet adhesion: At 1800 sec−1 shear rate, which approximates venous blood flow, addition of RMP increases the adhered mean aggregate size from 47 to 53 μm2 (p<0.03) but decreased the number of adhering objects from 1380 to 1242 (p<0.01). At lower (900 sec−1) or higher (2700 sec−1) rate, the RMP effects disappeared. (3) Two-color flow cytomrtry showed that RMP do not conjugate with single platelets but do with platelet micro-aggregates induced by ADP. When platelet micro-aggregates are diluted with PBS (1:10), they usually disaggregate rapidly (t1/2 = 10–15 min) but in presence of RMP, the rate of dissociation was much slower (t1/2 = 30–40 min). Conclusions:These results reveal that RMP can interact with weakly activated platelets to enhance platelet adhesion and aggregation and stabilize platelet aggregates. Since these effects were seen with heparinized but not with citrated blood, calcium may be a cofactor for this interaction. We suggest that RMP-platelet interaction could play a role in hemostasis, and that therapeutic RMP may improve hemostatic abnormality in thrombocytopenia and platelet dysfunction partly by this mechanism, augmenting the limited platelet function. Disclosures:No relevant conflicts of interest to declare.

Red Cell Microparticles (RMP) As Hemostatic Agent: Summary of Recent Advances

Abstract 2260 Introduction:Microparticles (MP) derived from platelets (PMP), endothelia (EMP) and leukocytes (LMP) have received much attention as biomarkers in cardiovascular and inflammatory disorders. However MP derived from red cells (RMP) have been less well investigated. We previously presented evidence for the efficacy of RMP as a hemostatic agent in vivo and other effects such as on thrombin generation. Here we report data on in vitro proxies of hemostatic potential of RMP which further support its expected value as a hemostatic agent, and provide clues about mechanism of action. Methods: (i)RMP were produced by high-pressure extrusion of washed RBC. (ii) The effect of RMP on coagulation/clotting was assessed by thromboelastography (TEG) using whole blood or factor-deficient plasmas tested in presence vs. absence of added RMP (1×108/mL). Patient samples included those with coagulopathy, thrombocytopenia, thrombocytopathy. (iii) The effect of RMP on platelet aggregation was assessed by aggregometry (Chrono-Log). (iv) The effect of RMP on shear-induced platelet adhesion was by cone-and-well device (Diamed IMPACT-R) yielding measures of mean aggregate size (AS), percent surface coverage (SC) and number of objects (OBJ). Results: (1)RMP produced as above express procagulant phospholipids as judged by 20–30% of them binding annexin V. (2) RMP increased procoagulance in all factor-deficient plasmas tested (FII, V, VII, VIII, IX, X, XI, XII, XIII). The most pronounced effects were observed with FII, FVIII, FIX, FX deficiency, most markedly, FVIII and IX. RMP give significant correction for deficiencies as low as 5–10% normal levels. (3) RMP augmented platelet aggregation induced by low-dose ADP (0.2 μM) or arachidonic acid (0.3 mM). RMP largely eliminated dissociation of platelet aggregates after low-dose ADP. (4) At shear rate of 1800 sec−1, which approximates venous flow, aggregate size (AS) was significantly increased (p=0.02). (5) Using flow cytometry, RMP were observed to interact with platelet microaggregates expressing markers of activation, but not with free resting platelets. (6) RMP partly or completely corrected abnormal parameters seen on TEG in blood from patients with thrombocytopenia (aplastic anemia, ITP) as well as platelet dysfunctions induced by ASA or Plavix. It also improved or corrected abnormal parameters in coagulopathies such as hemophilia A with low inhibitor; and anticoagulant therapy with wafarin, LMWH, or Dabigatran. Summary/Discussion:(1) Increased procoagulance in all factor deficient plasma by RMP indicates that RMP provide anionic surface for clotting factors. Since this was most pronounced with FII, FVIII, FIX, and FX, and was strongest in FVIII, FIX, it appears that RMP have some specificity for assembly of tenase. (2) Correction of clotting abnormality in thrombocytopenia and platelet dysfunction by RMP suggests that RMP may provide additional catalytic surface for coagulation, as well as contributing modestly to clot strength. The latter effect may depend on presence of weakly activated platelets. (3) The increased maximum amplitude (MA) seen in TEG by RMP suggests that RMP may enhance platelet-fibrin interaction, increasing clot stability. Taken together, these data support the potential use of RMP as a universal hemostatic agent. Disclosures:No relevant conflicts of interest to declare.

Ristocetin Exposes the ADAMTS13 Cleavage Site of Von Willebrand Factor and Enhances VWF Cleavage

AbstractAbstract 4317Ristocetin, a peptide antibiotic from the soil bacterium Nocardia lurida, has been used for decades as a tool to diagnose deficiency or dysfunction of von Willebrand factor (VWF) in von Willebrand disease. Ristocetin is able to assess the functional state of VWF because it induces the interaction of VWF with the platelet glycoprotein (GP) Ib-IX-V complex in the absence of shear stress or VWF immobilization, conditions normally required in vivo for their interaction. Presumably, ristocetin is able to do this by inducing an allosteric change in VWF that exposes the binding site for GPIbα. Ristocetin is one of two widely used modulators of the VWF–GPIb α interaction (the other being botrocetin), and the one that induces an interaction that most closely mimics shear-induced platelet adhesion and aggregation. Recently, Shim et al, (Blood, 2008;111(2):651-7) demonstrated that VWF bound to platelets was a better substrate for the plasma metalloprotease ADAMTS13, raising the possibility that exposure of the GPIbα binding site on VWF could be coupled to exposure of the ADAMTS13 cleavage site. Another possibility would be that the tensile force experienced by a VWF strand with multiple bound platelets in a shear field would be sufficient to stretch VWF and expose the ADAMTS13 cleavage site. We therefore evaluated whether ristocetin alone could enhance ADAMTS13 cleavage of VWF in the absence of shear force. We used four VWF sources for these experiments: plasma; purified, multimeric VWF from plasma; a recombinant fragment encompassing the three A domains (A1A2A3); and two recombinant A2 domains, one containing a previously identified ristocetin-binding site between D1459 and P1465, and the other lacking it. Ristocetin at 1.0 mg/ml induced the cleavage of VWF by ADAMTS13 in plasma or of the purified multimeric form as efficiently as did 1.5 M urea, the standard reagent and concentration used for this assay. Similarly, ristocetin accelerated cleavage of the monomeric A1A2A3 fragment. Finally, and somewhat surprisingly, ristocetin accelerated cleavage of the isolated A2 domain, but only when the D1459–P1465 sequence was included in the construct. Vancomycin, a related antibiotic, did not have this effect. Our data suggest that exposure of the ADAMTS13 cleavage site is not only induced by tensile force in vivo, but also by other more subtle biochemical forces. These findings also indicate that exposure of the binding site for GPIbα is coupled to exposure of the ADAMTS13 cleavage site in VWF, perhaps providing part of the explanation for why platelet-bound VWF is a better ADAMTS13 substrate and for why newly released ultralarge VWF is both capable of spontaneously binding platelets and of being cleaved rapidly by ADAMTS13 in the presence of minimal shear stress. Finally, our findings also suggest that ristocetin might be a more specific reagent to evaluate the activity of ADAMTS13 for cleaving multimeric VWF in vitro. Disclosures:No relevant conflicts of interest to declare.

Soluble Monomeric Von Willebrand Factor A1A2A3 (R1450E) Mutant Fragment Enhances Shear-Induced Platelet Aggregation and Platelet Adhesion to Fibrin(ogen) Via αIIbβ3 Under High Shear Stress.

Abstract 4025Poster Board III-961Recently, we described that the gain of function mutation R1450E in the A1 domain of von Willebrand factor (VWF) eliminates the formation of catch bond with glycoprotein (GP)Ibα, prolonging the bond lifetimes at low forces. Because those studies were performed with the mutant immobilized on a plastic surface, we further characterize the effect of this mutant on platelet function in solution and under shear stress. Both wild type (WT) and mutant A1A2A3 proteins were expressed in HEK293 cells and purified to homogeneity. The monomeric state of A1A2A3 proteins were assessed by gel filtration chromatography and neither of the proteins had formed dimers or any higher order aggregates. The recombinant A1A2A3 mutant bound spontaneously to GPIbα without the modulator ristocetin with a half-maximal binding observed at 65 ± 8 nM. This apparent dissociation constant was comparable to that of WT (50 ± 10 nM) in the presence of ristocetin. The mutant failed to induce spontaneous platelet aggregation under stirring conditions, and blocked 100% ristocetin-induced platelet agglutination (RIPA) at concentration of 250 nM. At the same concentration, the mutant increased shear-induced platelet aggregation (SIPA) at 500s-1 and 5000s-1 shear rates, reaching 42% and 66%, respectively, while SIPA did not exceed 18% in the presence of WT. The anti-αIIbβ3 antibody 7E3 blocked the effect of the mutant on SIPA. Blood was then incubated with the mutant (250 nM) and perfused over a surface coated with fibrin(ogen) at different shear rates. Blood containing WT resulted in <10% surface coverage by platelets after 1.5 minutes while platelets from blood containing the mutant rapidly bound covering 100% of the fibrin(ogen) surface area at 1500s-1. At shear rate of 2500s-1, surface coverage was 20% for the mutant and 0% for WT fragment. EDTA and antibodies 6D1 (GPIbα) and 10E5 (αIIbβ3) effectively blocked mutant-mediated platelet adhesion and thrombus formation under high shear rates. The addition of ristocetin (0.5 mg/ml) to whole blood prior perfusion reproduced the effect of the mutant. Here, we describe an A1A2A3 mutant that bound spontaneously to GPIbα but affected differently RIPA and SIPA. These results suggest that hydrodynamic forces directly act on the GPIbα-mutant A1A2A3 complex, regulating signaling. In addition, platelet activation induced by the binding of soluble mutant A1A2A3 or plasma VWF results in αIIbβ3-mediated platelet adhesion to fibrin(ogen) under high shear rates. Disclosures:No relevant conflicts of interest to declare.

Shear-induced interaction of platelets with von Willebrand factor results in glycoprotein Ibα shedding

Shear-induced platelet adhesion through the interaction of glycoprotein (GP) Ibalpha with von Willebrand factor (VWF) exposed at the injured vessel wall or atherosclerotic plaque rupture is a prerequisite for the physiological hemostatic process or pathological thrombus formation in stenosed arteries. Here we show that shear-induced interaction of platelets with immobilized VWF results in GPIbalpha ectodomain shedding. Washed platelets were exposed to VWF-coated glass capillary or cone-and-plate viscometer at different shear rates, and GPIbalpha ectodomain was shed from platelets, while a small mass of GPIbalpha COOH-terminal peptide, approximately 17 kDa, was increased correspondingly. The extent of GPIbalpha shedding was enhanced with the concentration of immobilized VWF and the time duration of constant shear stress, whereas it was obviously reduced with the decreased number of adherent platelets. Pretreatment of platelets with membrane-permeable calpain inhibitors and metalloproteinase inhibitor abolished shear-induced GPIbalpha shedding. Furthermore, GPIbalpha shedding was obviously diminished by anti-integrin-alphaIIbbeta3 monoclonal antibody SZ21, phosphatidylinositol 3-kinase inhibitor wortmannin, and cell-permeable calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. These results indicate that shear-induced platelet-VWF interaction results in calpain and metalloproteinase-dependent GPIbalpha ectodomain shedding. These findings not only have a physiological implication in understanding the presence of glycocalicin in normal circulation, but also suggest a novel mechanism for the negative regulation of platelet function and the limitation of platelet thrombus infinite formation under pathophysiological flow conditions.

Cross validation of aspirin effect in healthy individuals by Impact-R and PFA-100: A double blind randomized placebo controlled trial

The main objective of this study was to compare testing for aspirin response in healthy volunteers by two high shear methods in a randomized double blind placebo controlled study. Seventeen healthy male individuals were randomized for aspirin 160 mg per day for 7–10 days, and 20 age matched controls for placebo for the same period. At study entry and 7–10 days thereafter we determined high shear-induced platelet adhesion to polystyrene after pre-incubation with arachidonic acid using the Cone and Plate(let) analyzer (Impact-R), and the closure time of collagen/epinephrine cartridges obtained by the PFA-100 (CEPI-CT). Platelet adhesion to polystyrene after preincubation with arachidonic acid was median 3.7% (range 0.6–8.0) before study entry and median 6.7% (range 2.8–11.0) after 7–10 days of aspirin (p < 0.001). Changes were not significant in the placebo group. By the PFA-100 CEPI-CT was median 211 s (range 130–300 s) before aspirin, and 300 s in all individuals taking aspirin for 7–10 days (p < 0.001). Post-treatment data obtained by the Impact-R and PFA-100 were discordant in seven cases from the placebo group, and in one subject on aspirin. The response to aspirin varied considerably among healthy individuals, but both methods were suitable to demonstrate the aspirin effect. There was, however, a significant level of absent concordance between the tests. Since the trial design cannot provide data on the specificity of the different tests, only clinical experience can determine their usefulness.

Functional Impairment of Von Willebrand Factor in Hypertrophic Cardiomyopathy

Hypertrophic obstructive cardiomyopathy submits blood to conditions of high shear stress. High shear stress impairs von Willebrand factor (VWF) and promotes abnormal bleeding in aortic stenosis. We sought to evaluate VWF impairment and its relationships to baseline or exercise obstruction in hypertrophic cardiomyopathy (HCM). Outflow obstruction was evaluated by rest and exercise echocardiography in 62 patients with HCM (age 44+/-16 years, 40 males). HCM was considered obstructive in 28 patients with rest or exercise peak gradient >or=30 mm Hg. Blood was sampled to assess VWF. History of bleeding was recorded. Baseline median (25th to 75th percentile) peak gradient was 11 (5-62) mm Hg. Shear-induced platelet adhesion was impaired in patients with obstructive HCM. The ratio of VWF-collagen-binding activity to antigen and the percentage of high-molecular-weight multimers of VWF were lower in patients with obstructive HCM than in those with nonobstructive HCM (0.49 [0.43 to 0.59] versus 0.82 [0.73 to 1.03] and 5.0% [3.9% to 7.2%] versus 11.7% [10.8% to 12.5%], respectively; both P<0.0001). Platelet adhesion time, VWF-collagen-binding activity-to-antigen ratio, and the percentage of high-molecular-weight multimers correlated closely and independently with peak gradient (r=0.81, r=-0.68, and r=-0.89, respectively; all P<0.0001). According to receiver operating characteristic curves, a peak gradient threshold of 15 mm Hg at rest and 35 mm Hg during exercise was sufficient to impair VWF. Conversely, VWF function tended to improve with a decrease in peak gradient. Obstructive HCM patients had a trend toward abnormal spontaneous bleeding. In obstructive HCM, VWF impairment is frequent and is closely and independently related to the magnitude of outflow obstruction. A resting peak gradient of 15 mm Hg is sufficient to impair VWF. VWF abnormalities might favor abnormal bleeding in this setting.

Peptides Derived from Fibronectin Type III Connecting Segments Promote Endothelial Cell Adhesion but Not Platelet Adhesion: Implications in Tissue-Engineered Vascular Grafts

The development of a completely tissue-engineered small-caliber prosthesis suitable for incorporation into an in vivo vascular network is fraught with many challenges, including overcoming resistance to endothelialization and susceptibility to thrombogenesis. In this work, recombinant human fibronectin-derived low-molecular-weight peptide fragments were studied for their ability to promote cell type-specific alpha(4) integrin-mediated adhesion. Two populations of primary human endothelial cells were examined and found to express alpha(4) integrin receptors on their surfaces; on the contrary, human platelets were not found to be expressers of alpha(4) integrins. A peptide fragment isolated from the variably spliced human fibronectin type III connecting segment-1 (CS-1) domain was determined to mediate statistically significant endothelial cell alpha(4) integrin-mediated adhesion. In contrast, the fibronectin type III CS-1 fragment did not support human platelet adhesion under physiological fluid shear conditions, although fully intact human fibronectin molecules supported shear-induced platelet adhesion. This suggests that platelets bind to fibronectin in regions not encompassing the CS-1 domain. In conclusion, this work has demonstrated that the low-molecular-weight peptide CS-1 could serve as a cell-selective adhesion mediator in the engineering of a more-compatible small-caliber vascular graft lumen interface.

Mechanisms of Static and Shear-Induced Platelet Adhesion: Differences in the Adhesion Supported by MMRN1 Comparisons with Other β3 Integrin Ligands.

Platelet adhesion and aggregation at the site of vascular injury are key events in hemostasis and thrombosis. These processes are supported by interactions between platelet glycoprotein (GP) receptors (including integrin αIIbβ3, GP Ib-IX-V and GPVI) and ligands that include von Willebrand factor (VWF), collagen, and fibrinogen (Fg). Recently, the polymeric protein multimerin 1 (MMRN1) was identified to bind β3 integrins. Normally, MMRN1 is sequestered within secretion granules of platelets, megakaryocytes, and endothelium until its release. In static adhesion assays, MMRN1 supports platelet adhesion to integrins αIIbβ3 and αvβ3 by an RGD-dependent mechanism.Study goals and methods: To further determine the mechanisms of MMRN1 binding to platelets, we investigated (i) the importance of platelet activation in MMRN1 binding to platelets, (ii) the ability of MMRN1 to support platelet adhesion compared to other adhesive ligands (Fg, VWF), and (iii) the role of β3 integrins in MMRN1 binding to platelets at low and high shear.Results: The binding of secreted platelet MMRN1 to thrombin activated platelets was significantly reduced by antibody inhibitors of ligand binding to αIIbβ3 and αvβ3. Thrombasthenic platelets (GT), deficient in αIIbβ3 and αvβ3 integrins, stored normal quantities of MMRN1 but after activation, they retained 34% less MMRN1 than normal platelets. Platelet adhesion experiments confirmed that αIIbβ3/αvβ3 and other binding sites supported MMRN1 binding to platelets. MMRN1 supported platelet adhesion at both low (150 s-1) and high (1500 s-1) shear rates. Like platelet adhesion to VWF, platelet adhesion to MMRN1 was greater at high shear rates. Platelet stimulation by agonists was essential to induce platelet binding to MMRN1 in static and shear adhesion assays, but not to induce platelet adhesion to Fg and vWF. While platelets activated by ADP and TRAP showed similar adhesion to Fg and VWF, TRAP induced more platelet adhesion to MMRN1 at high shear rates than ADP. Inhibitors of ligand binding to αIIbβ3 and αvβ3 had greater effects on high compared to low shear platelet adhesion to MMRN1.Conclusions: These data indicate interesting differences in the mechanisms that support platelet adhesion to MMRN1 and other β3 ligands, which could be important for molecular events in hemostasis and thrombosis. The activation-dependent binding of MMRN1 to platelets, augmented by high shear flow, may reflect the unique recognition properties of platelet β3 integrins and/or exposure of other binding sites (e.g. phosphatidylserine) on activated platelets that promote adhesive interactions with MMRN1.

Strenuous, acute exercise affects reciprocal modulation of platelet and polymorphonuclear leukocyte activities under shear flow in men

Vigorous exercise transiently increases the risk of primary cardiac arrest. The reciprocal modulation of platelet and polymorphonuclear leukocyte (PMN) activities is important in the pathogenesis of thrombosis. This study investigates how strenuous, acute exercise affects platelet-PMN reciprocal modulation by closely examining 18 sedentary men who exercised strenuously on a bicycle ergometer. Shear-induced platelet activation, PMN interaction with surface-adherent platelets under shear flow, and PMN-dependent inhibition of platelet activation were measured both before and immediately after exercise. Analytical results can be summarized as follows: (i) shear-induced platelet adhesion on fibronectin-coated surface as well as ADP-induced release of platelet soluble P-selectin release and elevation of [Ca2+]i significantly increases after strenuous exercise; (ii) strenuous exercise is associated with higher velocity and percentage of rolling PMNs and lower numbers of PMNs remaining bound to surface-adherent platelets under shear flow than at rest; (iii) PMN-dependent inhibition of platelet [Ca2+]i elevation and soluble P-selectin release after strenuous exercise is much greater than that at rest; and (iv) strenuous exercise increases PMN-derived nitric oxide metabolite level and reduces oxidized low-density lipoprotein-promoted interaction between platelets and PMNs. Therefore, we conclude that platelet activity may be sensitized by strenuous exercise. However, strenuous exercise can also simultaneously enhance the antiplatelet effect of PMNs. The finding provides a new insight into the negative feedback of PMNs against exercise-evoked platelet-related thrombotic risk.

Platelets and von Willebrand factor

The interaction of platelets with von Willebrand factor (VWF) is crucial in the initiation of any hemostatic or thrombotic process. VWF enables the platelet, via its surface glycoprotein receptors, to adhere to exposed subendothelium and to respond to shear stress in the blood. Via VWF that is stored and released from platelet α-granules and from Weibel–Palade bodies of endothelial cells, the hemostatic system can respond locally to lesions in the vessel wall and can initiate the activation of other platelets. This review describes the molecular structure of VWF, its functions and its interactions with the platelet membrane glycoprotein receptors GP Ib–IX–V and GP IIb–IIIa. As well, the role of VWF in shear-induced platelet adhesion and aggregation is described, and mechanisms are discussed that control the size of VWF multimers and the responsiveness of platelets to multimeric VWF. Finally, the review discusses the role of locally released VWF from platelet α-granules and from Weibel–Palade bodies for platelet activation in neonates and adults.

Pharmacological approach to prevent high shear stress induced thrombus formation on titanium surface

BACKGROUND: Titanium-alloys (Ti) are widely used as blood biocompatible material for blood pumps and coronary stents. Thrombus formation on the Ti surface often results from high shear stress induced-platelet activation, and it is contributory to pump failure and coronary reocclusion, respectively. Although heparin and aspirin are routinely used to prevent thrombus formation in clinical settings, the efficacy is not satisfactory. The aim of this study was to elucidate the mechanisms of thrombus formation on Ti surface induced by high shear stress and to evaluate the efficacy of antithrombotic agents. METHODS: A cone-and-plate viscometer and a parallel flow chamber were used for shear-induced platelet aggregation and adhesion studies, respectively. Platelet microaggregation of citrate anticoagulated platelet rich plasma samples were measured by flow cytometry. Anti-GPIIIa antibody was used to identify the number of platelets adhering to the Ti surface after infusion of heparinized whole blood. RESULTS: Heparin (10 unit/ml) enhanced shear (108 dynes/cm2) induced human platelet aggregation by 22%. Absorptions of vWF and fibrinogen onto Ti surface were increased within 60 min after incubation with plasma and both plasma proteins enhanced platelet adhesion induced by high shear stress (111 dynes/cm2). Indomethacin (30 mM) had no effect on shear-induced platelet adhesion. Maximal concentration of TAK-029, a GPIIb/IIIa antagonist, prevented the platelet adhesions to non-, vWF- and fibrinogen-coated Ti surface by 89%, 75% and 98%, respectively. CONCLUSION: Treatment with a GPIIb/IIIa antagonist is an appropriate option to prevent shear-induced thrombus formation on Ti surface.

Shear-induced platelet activation and adhesion on human pulmonary artery endothelial cells seeded onto hydrophilic polymers.

We evaluated platelet activation and adhesion on two plasma polymerized surfaces, N-vinyl pyrrolidone (NVP) and gamma-butyro lactone (GBL), which have been shown previously to promote endothelial cell growth and adhesion as well as fibronectin-coated glass (1 microg/cm(2)) coverslips. Human pulmonary artery endothelial cells were seeded onto coverslips at a low density ( approximately 20,000 cells/cm(2)) and grown to confluence (3-5 days). The materials, both with and without ECs, were then exposed to a shear rate of 400 s(-1) in a closed loop recirculating flow system containing human platelet-rich plasma. Plasma samples were taken at 0, 5, 15, 30, and 60 min and analyzed for platelet and coagulation activation. The coverslips were examined for EC coverage and platelet adherence. EC retention over a 1-h period was approximately 75% for all three materials. All three materials without ECs were highly platelet activating having similar P-selectin expression, platelet factor 4 (PF4) release, mepacrine uptake, and microparticle production. Both microparticle production and platelet adhesion were significantly lower in EC-seeded materials. Dense granule and PF4 release were both slightly diminished in all three materials seeded with ECs. P-selectin expression was reduced slightly for GBL, but remained the same for the other two materials. The EC-seeded materials displayed favorable characteristics with respect to platelet activation and adhesion; however, they still demonstrated some thrombogenic tendencies due to EC loss and exposure of the underlying substrate. Therefore, both EC coverage and EC hemostatic function are important factors in determining the thromboresistance of an EC-seeded surface.

Characterization of Murine Anti-Glycoprotein Ib Monoclonal Antibodies That Differentiate between Shear-Induced and Ristocetin/Botrocetin-Induced Glycoprotein Ib-von Willebrand Factor Interaction

Platelet adhesion to vascular subendothelium under conditions of high shear stress is mediated by the platelet glycoprotein (GP) Ib-von Willebrand Factor (vWF) interaction. The aim of this study was to characterize the murine monoclonal antibodies (MoAbs) 27A10 and 28E6, both raised against purified GPIb. The MoAb 27A10 is a potent inhibitor of shear-induced platelet adhesion to collagen type I in a flow chamber at shear rates of 1,300 and 2,700 s^–1. 20 μg/ml of MoAb 27A10, furthermore, could completely block shear-induced aggregation in a modified Couette viscometer at shear rates of 1,000 and 4,000 s^–1. On the other hand, MoAb 27A10 had a negligible effect on botrocetin-induced GPIb-vWF binding and is only a poor inhibitor of the ristocetin-dependent interaction. In contrast, MoAb 28E6 did abolish both the ristocetin- and botrocetin-induced GPIb-vWF binding, whereas it did not block the shear-induced interaction. Thus, we identify here two anti-GPIb MoAbs 27A10 and 28E6 that either preferentially inhibit the shear-induced or the ristocetin/botrocetin-induced platelet-vWF interaction. With these tools it should be possible to more clearly define the mechanisms by which platelets bind to vWF in vivo.

Incorporation of an Asp-Ser Sequence to Form an RGDS-like Motif in Hirutonin: The Effect on In Vitro Platelet Function

We investigated the effect on in vitro platelet function of hirutonin, a modified hirutonin with an RGD-like motif, a pseudo-RGDS peptide and a linear RGDS peptide. Inhibition of expression of surface fibrinogen on ADP-activated platelets with 40 μM of the peptide was as follows: hirutonin 10±3%, modified chimeric peptide 26±5%, pseudo-RGDS 66±11% and linear RGDS 93±13%. Both hirutonin and the chimeric peptide significantly inhibited ADP-induced platelet activation as detected by CD62 expression. Unlike the RGDS and pseudo-RGDS controls, neither the chimeric peptide nor the parent hirutonin inhibited ADP-induced platelet aggregation even at 140 μM. The chimeric hirutonin peptide reduced ATP release from ADP-stimulated platelets by 40±4%. This inhibition was stronger than that caused by hirutonin (23±13%), but less than the RGDS (90±2%) and pseudo RGDS-peptides (59±11%). Primary platelet haemostasis was slightly but not significantly affected by the peptide at 40 and 80 μM. However, shear-induced platelet adhesion to vWF and especially subsequent aggregate formation was interrupted after the addition of the chimeric peptide. Similar results were obtained with hirutonin. This inhibition was not as marked as with the RGDS- and pseudo-RGDS peptides. Both the parent hirutonin and the chimeric peptide caused prolongation of the clinical coagulation assays aPTT and TT. In conclusion, the chimeric hirutonin peptide with introduction of the RGD motif retained its anticoagulant effect but had little formal disintegrin activity. Instead, it appeared to have novel anti-platelet effects that may be of therapeutic use.

OPTIMAL HEPARIN-PROTAMINE REVERSAL RATIO

Introduction: Some investigations have demonstrated an anti-platelet effect of excess protamine, [1] while others have suggested that the usual doses of protamine fail to reverse the anti-platelet effects of heparin. [2] We have previously reported use of the Clot Signature Analyzer (CSA[registered sign]) to assess the anti-platelet activity of protamine. Here we report use of the CSA[registered sign] to assess the effect of heparin reversed with different protamine doses on platelet function. The CSA[registered sign] tests whole blood shear-induced platelet adhesion (the platelet-induced hemostasis time, PHT), collagen-induced thrombus formation (CITF) and the clotting time (CT) in conditions mimicking physiologic blood flow. Methods: After HIC approval, paired whole blood 3ml samples from 9 volunteers were drawn into syringes containing 240[micro sign]l PBS or 120[micro sign]l heparin 1 mg/ml and 120[micro sign]l protamine 1 mg/ml (HEP/PROT 1/1). All samples were incubated for 90 seconds and assayed in the CSA[registered sign]. A similar analysis was performed comparing 120[micro sign]l heparin 1 mg/ml "reversed" with 180[micro sign]l protamine 1 mg/ml (HEP/PROT 1/1.5) with paired controls and 120[micro sign]l heparin 1 mg/ml "reversed" with 240[micro sign]l protamine 1 mg/ml (HEP/PROT 1/2) compared with 120[micro sign]l heparin 1 mg/ml "reversed" with 180[micro sign]l protamine 1 mg/ml (HEP/PROT 1/1.5). Statistical analysis was with the Wilcoxon signed rank test, significance taken at a P value < 0.05 and values expressed as mean +/- SEM. Results: HEP/PROT 1/1: The PHT, CITF and CT were prolonged significantly by heparin/protamine 1/1 incubation compared with controls (Figure 1, marked *). HEP/PROT 1/1.5: The PHT, CITF were not significantly prolonged with heparin/protamine 1/1.5 incubation compared with controls (Figure 2). Only the CT was significantly prolonged (Figure 2). HEP/PROT 1/1.5 v HEP/PROT 1/2: The PHT and CITF were significantly prolonged by heparin/protamine 1/2 compared to heparin/protamine 1/1.5 (Figure 3).Figure 1Figure 2Figure 3Discussion: 1:1 protamine "reversal" still allows an anti-platelet effect of heparin, as shown by a significantly prolonged PHT, CITF and CT. Increasing the protamine "reversal" dose to 1:1.5 successfully reverses heparin's effect on the PHT and CITF, with CT modestly prolonged, suggesting this approximates the optimal reversal ratio for preservation of in vivo platelet function. Further increase in the heparin/protamine ratio to 1/2 induces an increase in the PHT and CITF, possibly attributable to excess protamine. The mechanism of this protamine effect warrants further investigation. We conclude that protamine at 1.5x the usual reversal dose optimally antagonizes the anti-platelet effects of heparin, while minimizing the intrinsic anti-platelet effects of protamine.