Support Platelet Adhesion Research Articles

C57BL/6JOlaHsd Mice with Tandem Deletion of the Multimerin 1 and Alpha-Synuclein Genes Have Impaired Platelet Function in Vivo and in Vitro That Can Be Corrected by Multimerin 1

Studies of mice with genetic deficiencies have provided important insights on the functions of many proteins in thrombosis and hemostasis. Recently, a strain of mice (C57BL/6JOlaHsd, an inbred strain of C57BL/6J) has been identified to have a spontaneous, tandem deletion of the multimerin 1 and α-synuclein genes, which are also adjacent genes on human chromosome 4q22. Multimerin 1 is an adhesive protein found in platelets and endothelial cells while α-synuclein is a protein found in the brain and in blood that is implicated in neurodegenerative diseases and exocytosis. In vitro, multimerin 1 supports platelet adhesion while α-synuclein inhibits α-granule release. We postulated that the loss of multimerin 1 and α-synuclein would alter platelet function and that recombinant human multimerin 1 might correct some of these abnormalities. We compared platelet adhesion, aggregation and thrombus formation in vitro and in vivo in C57BL/6JOlaHsd and C57BL/6 mice. Thrombus formation was studied by using the ferric-chloride injured mesenteric arteriole thrombosis model under intravital microscopy. We found that platelet adhesion, aggregation and thrombus formation in C57BL/6JOlaHsd were significantly impaired in comparison to control, C57BL/6 mice. The number of single platelets, deposited 3–5 minutes after injury, was significantly decreased in C57BL/6JOlaHsd mice (P <0.05, platelets/min: C57BL/6 = 157 ± 15, n=16; C57BL/6JOlaHsd = 77 ± 13, n=17). Moreover, thrombus formation in these mice was significantly delayed. Thrombi in C57BL/6JOlaHsd were unstable and easily dissolved, which resulted in significant delays (P<0.001) in vessel occlusion (mean occlusion times: C57BL/6 = 15.6 ± 1.2 min, n=16; C57BL/6JOlaHsd = 31.9 ± 2.1 min, n=17). We further tested platelet function in these mice by ADP and thrombin induced platelet aggregation using platelet rich plasma and gel-filtered platelets, respectively. Although no significant differences were seen with ADP aggregation, thrombin-induced platelet aggregation was significantly impaired in C57BL/6JOlaHsd mice. Platelet adhesion to type I collagen (evaluated using microcapillary chambers, perfused at 1500 s−1 with whole blood) was also impaired in C57BL/6JOlaHsd mice. However, platelets from C57BL/6JOlaHsd mice showed a normal pattern of agonist-induced release of α-granule P-selectin. Multimerin 1 corrected the in vitro aggregation and adhesion defects of C57BL/6JOlaHsd platelets. Furthermore, the transfusion of multimerin 1 into C57BL/6JOlaHsd mice corrected the impaired platelet deposition and thrombus formation in vivo. No significant difference was found in tail bleeding time between the two groups of mice. As α-synuclein knockout mice have a shortened time to thrombus formation (Circulation 2007; 116:II_76), the effects of multimerin 1 on impaired platelet function in C57BL/6JOlaHsd mice provide supportive evidence that multimerin 1 contributes to platelet adhesion and thrombus formation at the site of vessel injury. The findings suggest multimerin 1 knockout mice will be useful to explore platelet function. The first two authors and participating laboratories contributed equally to this study.

Staphylococcal Superantigen-Like 5 Activates Platelets, and Supports Platelet Adhesion Under Flow Conditions, Which Is Mediated by GPIb- Alpha and Alpha-IIb-Beta-3

Introduction - Staphylococcal superantigen-like 5 (SSL5) is an exoprotein secreted by Staphylococcus Aureus and has been shown to inhibit neutrophil rolling over activated endothelial cells via a direct interaction with P-selectin glycoprotein ligand 1 (PSGL-1). Since PSGL-1 shows close homology to platelet glycoprotein Ib-alpha (GPIb), and as platelets have been shown to play an important role in neutrophil chemotaxis, we investigated whether SSL5 also interacts with platelets.Results - When purified recombinant SSL5 was added to washed platelets in an aggregometry set-up, full and irreversible aggregation was observed even in the absence of exogenous fibrinogen. Proteolysis of the extracellular part of GPIb-alpha or the addition of dRGDW abrogated platelet aggregation. SSL5 was shown to interact with glycocalicin, a soluble GPIb-alpha fragment, and binding of SSL5 to platelets resulted in GPIb-mediated signal transduction as evidenced by translocation of 14-3-3zeta to the actin cytoskeleton. When a mixture of isolated platelets and red cells was perfused over immobilized SSL5 at a shear rate of 300 s−1, large stable platelet aggregates were observed. Platelet adhesion was substantially reduced by proteolysis of GPIb-alpha or addition of dRGDW. In addition, real-time video analysis showed that initial tethering of the platelets was abrogated by proteolysis of GPIb-alpha. Furthermore, when whole citrated blood was perfused over immobilized SSL5, real-time video analysis revealed that platelets activated with the PAR-1 activating peptide SFLLRN made transient contacts with the immobilized SSL5, although no permanent contacts were observed. Finally, SSL5 was shown to interact with endothelial cell matrix (ECM) from cultured human umbilical vein endothelial cells. The interaction of SSL5 with ECM enhanced aggregation of platelets from whole blood to this ECM.Discussion - Here, we show that SSL5 is capable of activation, adhesion and aggregation of platelets, in which both GPIb-alpha and alpha-IIb-beta-3 play a role. We hypothesize that platelet activation by SSL5 could be important in colonization of the vascular bed and evasion of the immune system.

Redundant Mechanism of Platelet Adhesion to Laminin and Collagen under Flow: INVOLVEMENT OF VON WILLEBRAND FACTOR AND GLYCOPROTEIN Ib-IX-V

Although the role of collagen in thrombosis has been extensively investigated, the contribution of other extracellular matrices is still unclear. We have recently reported that laminin stimulates platelet spreading through integrin alpha(6)beta(1)-dependent activation of the collagen receptor glycoprotein (GP) VI under static condition. Under physiological high and low shear conditions, platelets adhered to laminin, and this was strongly inhibited by an antibody that blocks association between GPIb-IX-V and von Willebrand factor (VWF). Moreover, platelets of type III von Willebrand disease or Bernard-Soulier syndrome adhered to laminin at a low shear condition but not at a high shear condition. The specific binding of laminin to VWF was confirmed by surface plasmin resonance spectroscopy (BIAcore). These findings suggest that laminin supports platelet adhesion depending on the interaction of VWF and GPIb-IX-V under pathophysiological high shear flow. This mechanism is similar to that of collagen. We propose that integrins, GPVI, GPIb-IX-V, and VWF represent a general paradigm for the interaction between platelets and subendothelial matrices.

A new feasible method for fibrinogen purification based on the affinity of Staphylococcus aureus clumping factor A to fibrinogen

Plasma fibrinogen participates in several physiological and pathological events thus becoming a useful studying material in biomedical research. Here we report a new convenient method for fibrinogen purification based on the affinity of Staphylococcus aureus clumping factor A to fibrinogen. Clumping factor A (ClfA) is a cell wall-anchored surface protein of S. aureus bacteria that binds with a high affinity to the fibrinogen γ chain C-terminus via a segment encompassing the residues 221–550. This activity of ClfA (ClfA 221–550) was produced in fusion to the C-terminus of glutathione– S-transferase (GST) with recombinant technology and used as an affinity ligand to capture plasma fibrinogen. GST–ClfA 221–550 fusion protein was immobilized onto the glutathione-conjugated beads packed in a plastic column by its GST part. Then, this affinity column was loaded with citrated and heparinized human plasma. After washing out unbound proteins, column-captured fibrinogen was specifically eluted down with a citrate buffer solution (50 mM, pH 5.6). Purified human fibrinogen exhibited the ability to support platelet adhesion and aggregation and formed fibrin clot by thrombin, indicating that ClfA 221–550-purified human fibrinogen is a functionally active product. We also found that both the rat and mouse fibrinogens could be purified as well as human fibrinogen with this method. By virtue of its simplicity and feasibility, ClfA 221–550-based method would be very useful to the investigators who need fibrinogen to perform their studies.

Protein C supports platelet binding and activation under flow: role of glycoprotein Ib and apolipoprotein E receptor 2.

Activated protein C (APC) regulates thrombin generation and inhibits apoptosis. Endothelial protein C receptor (EPCR)-bound protein C is activated by thrombomodulin-bound thrombin. APC inactivates coagulation factors (F)Va/VIIIa and generates cytoprotective signaling downstream of protease-activated receptor-1 (PAR-1). Binding of APC to EPCR both modifies and induces PAR-1 signaling, but it is unknown if protein C interacts with cells in an alternative manner. To determine whether platelets possess receptors for protein C that can generate intracellular signals. Immobilized protein C or APC supported platelet adhesion, lamellipodia formation and elevation of intracellular Ca(2+). Adhesion of platelets to protein C or APC was inhibited by soluble recombinant apolipoprotein E receptor 2' (ApoER2') and by receptor-associated protein (RAP), an inhibitor of the low-density lipoprotein receptor family. Under shear, surface-bound protein C supported platelet adhesion and aggregation in a glycoprotein (GP)Ibalpha-dependent manner, and adhesion of platelets to immobilized protein C was abrogated by the addition of soluble forms of ApoER2' or RAP. APC bound to purified recombinant ApoER2' or GPIbalpha. Our data demonstrate that activation of platelets with rapid intracellular signaling caused by binding to immobilized protein C or APC occurs via mechanisms that require ApoER2 and GPIbalpha and that APC directly binds to purified ectodomains of the receptors ApoER2 and GPIbalpha. These findings imply that protein C and APC may directly promote cell signaling in other cells by binding to ApoER2 and/or GPIbalpha.

Platelet Dysfunction and a High Bone Mass Phenotype in a Murine Model of Platelet-Type von Willebrand Disease

The platelet glycoprotein Ib-IX receptor binds surface-bound von Willebrand factor and supports platelet adhesion to damaged vascular surfaces. A limited number of mutations within the glycoprotein Ib-IX complex have been described that permit a structurally altered receptor to interact with soluble von Willebrand factor, and this is the molecular basis of platelet-type von Willebrand disease. We have developed and characterized a mouse model of platelet-type von Willebrand disease (G233V) and have confirmed a platelet phenotype mimicking the human disorder. The mice have a dramatic increase in splenic megakaryocytes and splenomegaly. Recent studies have demonstrated that hematopoetic cells can influence the differentiation of osteogenic cells. Thus, we examined the skeletal phenotype of mice expressing the G233V variant complex. At 6 months of age, G233V mice exhibit a high bone mass phenotype with an approximate doubling of trabecular bone volume in both the tibia and femur. Serum measures of bone resorption were significantly decreased in G233V animals. With decreased bone resorption, cortical thickness was increased, medullary area decreased, and consequently, the mechanical strength of the femur was significantly increased. Using ex vivo bone marrow cultures, osteoclast-specific staining in the G233V mutant marrow was diminished, whereas osteoblastogenesis was unaffected. These studies provide new insights into the relationship between the regulation of megakaryocytopoiesis and bone mass.

Diagnosis and treatment of von Willebrand disease: new perspectives and nuances

In 1926, Erik von Willebrand, a Finnish internist andacademic, evaluated and described a 5-year-old girlwith extreme bleeding and bruising due to what hadbeen designated the A˚landic haemorrhagic diseaseby inhabitants of this archipelago island in the Gulfof Bothnia. Hjordis, the propositus, was the ninth of12 children born to a pedigree in which four femalesiblings had already died before the age of 4 withuncontrolled haemorrhage and in which 23 of 66family members, predominantly females, had expe-rienced significant bleeding and bruising complica-tions [1]. In fact, Hjordis herself eventually died atthe age of 13 during her fourth menstrual cycle.Professor von Willebrand mistakenly concluded thatthis bleeding diathesis was an unusual form ofhaemophilia and decided to label the new diseaseas pseudo-haemophilia to differentiate it from thesex-linked recessive haemophilia A. Little did vonWillebrand realize that this disease, eventually tobecomeeponymous[vonWillebranddisease(VWD)],would become the most commonly diagnosed con-genital bleeding disorder, with a prevalence rangingbetween 1 per 10 000 individuals to 1.3% [2]. Type1 VWD is the most common subtype, representingup to 75–80% of all cases while the subtype 3 VWDoccurs in approximately 1 per million population inthe United States and Europe [2]. Determination ofthe exact prevalence of VWD is hindered somewhatby the heterogeneity of clinical and diagnosticlaboratory features. Even Professor von Willebrandappreciated the challenges of diagnosing VWD as hecollaborated with Professor Rudolf Ju¨gens at theBerlin University to examine patients blood sampleswith a newly invented kapilla¨rtrombometer appa-ratus [3]. Although they were technically mistakenwhen they attributed the bleeding manifestations ofVWD to a platelet defect, their observations wereremarkably prescient since it was not until early1970s that the existence of a specific von Willebrandfactor (VWF) glycoprotein separate from factor VIIIwas finally appreciated and demonstrated to supportplatelet adhesion to the subendothelial matrix ofdamaged blood vessels.Over the last 81 years since von Willebranddescribed the bleeding disorder, there have beennumerous attempts and approaches to refine thediagnosis of the disease so that appropriate andefficacious treatment can be delivered. The clinicalphenotype of VWD includes, in part, information onwhether bleeding is spontaneous or related to surgi-cal or physical trauma; family history and inheri-tance pattern; menstrual history in women; age ofonset of bleeding (to distinguish between acquired vs.inherited coagulation disorders); and sites of bleed-ing(mucocutaneousvs.visceral,intra-articular,intra-muscular, or soft tissue locations). Subsequentlaboratory testing is necessary to exclude or confirmthe diagnosis and to further classify the subtype ofVWD. This article will focus on selected vagariesassociated with the ability to utilize clinical pheno-typic information gathered through the history andphysical examination to predict the presence ofVWD. The clinical diagnosis of VWD must be based,however, on more than physician suspicion andintuition. Thus, the need for confirmatory testing inthe laboratory. The frailties of conventional labora-tory testing for diagnosing VWD and the use of anin vitro surrogate of the bleeding time will also be

Regulation of Platelet Functions by Targeting STAT3 with a g-Quartet Oligonucleotide.

Collagen exposed on the subendothelial matrix supports platelet adhesion and aggregation at the site of vessel injury. Its interaction with the platelet membrane receptor GP V/FcRγ chain initiates intracellular signals that recruit the hematopoietic tyrosine kinase Syk to immunoreceptor tyrosine activation motifs (ITAMs) within the FcRγ cytoplasmic tail, leading a cascade of intracellular signal events that eventually activate platelets. In the present study, we demonstrated that collagen and the collagen-related peptide (CRP) stimulation also dose-dependently (2.5–10 mg/ml, 10 min) phosphorylated the signal transducers and activators of transcription 3 (STAT3) at the tyrosine residue 705 (Tyr705) in platelets, indicating that STAT3 participates in collagen-induced platelet activation, likely through a non-transcriptional mechanism. To test this hypothesis, we determined whether a STAT3-blocking G-rich oligonucleotide (T40214, GGGCGGGCGGGCGGGC) blocks platelet activation and aggregation induced by collagen or CRP. T40214 was designed based on G-rich DNA sequences, which were originally identified in the telomeres DNA to have the ability to form the protein-binding rigid G-quartet structures. It was formed through four guanine (G) bases into cyclic Hoogsteen H-bonding. NMR determined the tertiary structure of T40214 as two stacked identical G-quartets in the center and two G-C-G-C loop domains on the top and bottom, respectively. It has previously been shown to inhibit STAT3 phosphorylation in cancer cell lines and to induce tumor apoptosis in vivo. T40214 was delivered into platelets using nano-particle polyethylenimine (PEI) as the carrier. The treatment of washed platelets with T40214 inhibited STAT3 phosphorylation induced by collagen (10 mg/ml, 10 min), while the scramble oligonucleotides did not. Incubation of washed platelets and platelets-rich plasma (PRP) with T40214 inhibited collagen-induced platelets aggregation. Finally, thrombus formation on immobilized collagen was significantly reduced when whole blood treated with T40214 (10ug/ml) under a wall shear stress of 30 dyn/cm2. Results demonstrate a novel role of STAT3 in regulating the collagen-induced intracellular signal and platelet functions. The STAT3-blocking G-quartet offers a new class of potential platelet-regulating reagents derived from screening tertiary structure of the G-rich DNA sequences that form protein-binding G-quartets.

Distinct Functional Conformations of von Willebrand Factor A1 Domain in Support of Platelet-Surface or Platelet-Platelet Interactions.

The adhesive protein, von Willebrand factor (VWF), is generally considered a key substrate for platelet adhesion to the vessel wall, yet its role in platelet cohesion (aggregation) may be equally important for normal thrombus formation. In either case, the function of VWF is mediated by the primary interaction of the VWF A1 domain (VWF-A1) with glycoprotein (GP) Ibα, a component of the GPIb-IX-V receptor complex on the platelet membrane. Because normal plasma VWF in solution and GPIb coexist in circulating blood without any appreciable interaction, it has been postulated that conformational changes occur when VWF becomes immobilized and/or under the effect of pathologically elevated shear stress, such that binding to the receptor becomes possible and resultis in platelet tethering to a surface and shear-induced aggregation. Changes of the molecular shape of VWF, from coiled to extended, have been shown under the effect of hemodynamic forces, but evidence for conformational changes within VWF-A1 has remained elusive. The crystal structure of VWF-A1 in complex with a GPIbα amino terminal fragment has revealed that the VWF-A1 residues involved in the interaction are comprised between positions 544–614 and, in particular, do not include several positively charged Arg and Lys residues located in helices α4 and 5 (residues 627–668). The latter appear as likely candidates to interact with negatively charged residues in GPIbα as a consequence of potential conformational changes induced by tensile stress on the bond following an initial ligand-receptor contact. We tested this hypothesis by evaluating the ability of selected VWF-A1 mutants to support platelet adhesion or aggregation, respectively, under controlled flow conditions.Methods. We expressed in insect cells and purified a series of VWF-A1 fragments comprising residues 445–733. One fragment had native sequence and 8 had single or multiple substitutions of positively charged amino acid residues in helices α4 and/or α5. None of the substituted residues contribute to contacts with GP Ibα in the known crystal structures of the corresponding complex, and all except one were between 8 and 20 angstroms away from the closest GPIbα residue. All the fragments were dimeric (d) owing to the presence of interchain disulfide bond(s).Results: Native dVWF-A1 in solution supported platelet aggregation in a laminar flow field. Of the 8 mutants, 5 had variably decreased function (up to 95% less aggregation) and 2 had increased function (up to 200% increase in aggregation). The same results were observed with platelet-rich plasma in suspension or by measuring platelet aggregate formation with blood cells perfused over immobilized VWF-A1 at wall shear rates as high as 10,000 1/s. In contrast, as judged by the number of tethered platelets and their rolling velocities, all mutants supported adhesion as well as or better that the native VWFA-1 at all shear rates tested (500–25,000 1/s).Conclusions: These results provide structural evidence for the existence of different VWF-A1 conformers that can modulate adhesive properties with distinct effects on platelet adhesion to a surface or platelet aggregation.

Control of Platelet Adhesion by Rigidity Sensing at the Surface of Fibrin Clot.

Thrombus formation at sites of vascular injury must occur quickly to reduce blood loss, but is carefully controlled to limit vessel occlusion. Arrest of bleeding is mediated by adhesion and aggregation of platelets and the formation of the fibrin clot. While the interactions responsible for platelet adhesion and thrombus growth have been extensively researched, the mechanisms that limit platelet adhesion are not clear. We have previously demonstrated that plasma fibrinogen is a potent inhibitor of integrin-mediated leukocyte adhesion to fibrin clots and surface-bound fibrinogen, and have provided evidence that fibrinogen reduces cell adhesion by binding to the surface of fibrin rather than blocking leukocyte integrins. Accordingly, cells that engage fibrinogen molecules loosely bound to fibrin (soft substrate) are not able to consolidate their grip on the surface; subsequently, cells detach. Conversely, cells that adhere to the naked fibrin clot (rigid substrate) adhere firmly. Since fibrin and immobilized fibrinogen support platelet adhesion, we examined the effect of soluble fibrinogen on integrin αIIbβ3-mediated adhesion. We show that the anti-adhesive fibrinogen layer formed on the surface of fibrin inhibits platelet adhesion. We also demonstrate that fibrinogen immobilized on plastic at high densities (>20 μg/ml) supports weak platelet adhesion whereas at low concentrations (∼2 μg/ml) it is highly adhesive. An investigation of the mechanism underlying differential platelet adhesion indicates that platelet adhesion to rigid substrates (low-density fibrinogen and naked fibrin gel) induces much stronger phosphorylation of FAK and Syk kinases than that to soft substrates (high-density fibrinogen and fibrin exposed to soluble fibrinogen). Furthermore, the rigid, but not the soft substrates induce recruitment of signaling molecules talin and skelemin to αIIbβ3-containing focal adhesions. Consistent with their limited ability to induce sufficient signaling, soft substrates do not support platelet spreading. These data suggest that circulating fibrinogen prevents stable platelet adhesion by modifying the mechanical properties of the fibrin clot's surface which results in reduced force generation and insufficient signaling.

Abstract 1007: Platelet Glycoprotein VI (GPVI) Deletion Reduces Myocardial Infarction in Mice

Fibrillar collagen, exposed by damage of vascular endothelium, is the most thrombogenic component of the subendothelium, which, in addition to supporting platelet adhesion, is also a potent platelet activator. Platelet glycoprotein VI (GPVI) plays a major role in collagen-induced platelet activation, leading to thrombus formation. Inhibition of GPVI function may be a novel anti-platelet therapy with minimal bleeding risk. Recent evidence in FcRγ-chain-knockout (KO) mice, which also lack GPVI, shows attenuated myocardial infarction after ischemia and reperfusion. Unlike GPVI, which is exclusively expressed in platelets and megakaryocytes, FcRγ-chain also exists in white blood cells and immune cells. Thus, it is unclear whether GPVI deletion is cardioprotective. In this study, we compared myocardial infarction in GPVI-KO and age-matched wild-type (WT) mice. In parallel FcRγ-chain-KO mice were compared with age-matched WT mice. A major branch of left coronary artery was ligated for 30 min followed by reperfusion for 24 hrs. Myocardial infarction was measured by tetrozolium staining. Hemodynamics were not statistically different among the groups. Risk zone sizes were also similar between WT and GPVI-KO mice (0.020 ± 0.004 cm 3 and 0.022 ± 0.005 cm 3 , respectively) (Mean ± SD). However, percentage infarction of the risk zone was significantly smaller in GPVI-KO mice (averaged 22 ± 8 % vs. 45 ± 18 % in WT). Comparable protection was observed in FcRγ-chain-KO mice (12 ± 6 % vs. 46 ± 13 % in WT). Significant exposure of vascular collagen in the ischemia/reperfusion region of myocardium was revealed by FITC-labeled recombinant GPVI protein, injected prior to ischemia, indicating endothelium injury. Furthermore, there was significantly less expression of P-selectin, a marker of activated platelets, in the myocardium from GPVI-KO and FcRγ-KO mice when compared with WT mice. It is concluded that deletion of GPVI reduces myocardial infarction induced by ischemia and reperfusion, likely due to the attenuation of the platelet-collagen interaction. With its known anti-thrombotic effect and minimal risk of bleeding in animals, anti-GPVI therapy may offer a promising novel treatment for cardiovascular diseases.

Role of Streptococcus gordonii Surface Proteins SspA/SspB and Hsa in Platelet Function

Streptococcus gordonii colonization of damaged heart surfaces in infective endocarditis is dependent upon the recognition of host receptors by specific bacterial surface proteins. However, despite several attempts to identify the mechanisms involved in this interaction, the nature of the bacterial proteins required remains poorly understood. This study provides clear evidence that several S. gordonii surface proteins participate in the interaction with platelets to support platelet adhesion and induce platelet aggregation. S. gordonii strains were found to support strong (DL1-Challis, SK12, SK184, and Blackburn) or moderate (UB1545 delta hsa and CH1-Challis) adhesion or failed to support platelet adhesion (M5, M99, and Channon). In addition, under flow conditions, platelets rolled and subsequently adhered to immobilized S. gordonii at low shear (50 s(-1)) in an Hsa-dependent manner but did not interact with S. gordonii DL1 at any shear rate of >50 s(-1). S. gordonii strains either induced (DL1-Challis, SK12, SK184, UB1545 delta hsa, and M99) or failed to induce (M5, CH1-Challis, Channon, and Blackburn) platelet aggregation. Using a proteomic approach to identify differential cell wall protein expression between aggregating (DL1) and nonaggregating (Blackburn) strains, we identified antigen I/antigen II family proteins SspA and SspB. The overexpression of SspA or SspB in platelet-nonreactive Lactococcus lactis induced GPIIb/GPIIIa-dependent platelet aggregation similar to that seen with S. gordonii DL1. However, they failed to support platelet adhesion. Thus, S. gordonii has distinct mechanisms for supporting platelet adhesion and inducing platelet aggregation. Differential protein expression between strains may be important for the pathogenesis of invasive diseases such as infective endocarditis.

Platelet adhesion to dimeric beta-glycoprotein I under conditions of flow is mediated by at least two receptors: glycoprotein Ibalpha and apolipoprotein E receptor 2'.

The major antigen implicated in the antiphospholipid syndrome is beta2-glycoprotein I (beta2GPI). Dimerized beta2GPI binds to apolipoprotein E receptor 2' (apoER2') on platelets and increases platelet adhesion to collagen under conditions of flow. To investigate whether the interaction between dimerized beta2GPI and platelets is sufficiently strong to resist shear stresses. We studied the interaction of platelets with immobilized dimerized beta2GPI under conditions of flow, and further analyzed the interaction using surface plasmon resonance and solid phase immunoassays. We found that dimerized beta2GPI supports platelet adhesion and aggregate formation under venous flow conditions. Adhesion of platelets to dimerized beta2GPI was completely inhibited by the addition of soluble forms of both apoER2' and GPIbalpha, and the addition of receptor-associated protein and the removal of GPIbalpha from the platelet surface. GPIbalpha co-precipitated with apoER2', suggesting the presence of complexes between GPIbalpha and apoER2' on platelet membranes. The interaction between GPIbalpha and dimeric beta2GPI was of intermediate affinity (Kd = 180 nM) and Zn2+, but not Ca2+-dependent. Deletion of domain V from dimeric beta2GPI strongly reduced its binding to both GPIbalpha and apoER2'. Antibodies that inhibit the binding of thrombin to GPIbalpha inhibited platelet adhesion to dimeric beta2GPI completely, while antibodies blocking the binding of von Willebrand factor to GPIbalpha had no effect. Dimeric beta2GPI showed reduced binding to low-sulfated GPIbalpha compared to the fully sulfated form. We show that platelets adhere to dimeric beta2GPI under both arterial and venous shear stresses. Platelets adhere via two receptors: GPIbalpha and apoER2'. These receptors are present in a complex on the platelet surface.

A single high-affinity binding site for von Willebrand factor in collagen III, identified using synthetic triple-helical peptides

AbstractThe essential event in platelet adhesion to the injured blood vessel wall is the binding to subendothelial collagen of plasma von Willebrand factor (VWF), a protein that interacts transiently with platelet glycoprotein Ibα (GPIbα), slowing circulating platelets to facilitate firm adhesion through collagen receptors, including integrin α2β1 and GpVI. To locate the site in collagen that binds VWF, we synthesized 57 overlapping triple-helical peptides comprising the whole triple-helical domain of collagen III. Peptide no. 23 alone bound VWF, with similar affinity to that of native collagen III. Immobilized peptide no. 23 supported platelet adhesion under static and flow conditions, processes blocked by an antibody that prevents collagen from binding the VWF A3 domain. Truncated and alanine-substituted peptides derived from no. 23 either strongly interacted with both VWF and platelets or lacked both VWF and platelet binding. Thus, we identified the sequence RGQOGVMGF (O is hydroxyproline) as the minimal VWF-binding sequence in collagen III.

Characterization of the Mechanism of Interaction between C-Reactive Protein and GPIIb/IIIa.

C-reactive protein (CRP) is a well established marker for inflammation, and a good predictor of coronary heart disease. It is also known to interact with the platelet FcγRIIa and to enhance the inhibition of platelet aggregation by aspirin by an unknown mechanism. CRP has also recently been demonstrated to compete for PAC-1 binding in collagen stimulated platelets, suggesting that CRP interacts with GPIIb/IIIa. In order to study the mechanism of interaction with platelets directly, we carried out platelet adhesion assays. We coated plates with recombinant CRP which was in the native pentameric form as confirmed by size exclusion chromatography. Platelets adhered to immobilized CRP and to immobilized fibrinogen to a similar extent. Adhesion to CRP and fibrinogen was inhibited by GPIIb/IIIa antagonists but not by antibody to the FcγRIIa (IV.3). Platelet adhesion to CRP was increased 5-fold when platelets were treated with 1 mM MnCl2. Adhesion of MnCl2 treated platelets was also inhibited by GPIIb/IIIa antagonists. When viewed by confocal microscopy, the adherent platelets displayed pseudopodia, but did not spread fully. Treatment of platelets with MnCl2 stimulated lamellipodia formation and full platelet spreading on CRP. Analysis of the exposed residues on the surface of the CRP crystal structure identified two RGD-like sequences, RQD and DGK. The peptides KRQDN and VDGKP derived from CRP inhibited platelet adhesion to fibrinogen suggesting that these sequences could be responsible for the interaction with GPIIb/IIIa. Our data demonstrates that CRP in the native pentameric form interacts with GPIIb/IIIa and that it has an increased affinity for the activated conformation of GPIIb/IIIa. This interaction is likely to be due to an interaction with the surface exposed KRQDN and VDGKP CRP sequences. We have previously demonstrated that CRP acts as a GPIIb/IIIa antagonist when in solution. Furthermore, pentameric CRP cannot support platelet adhesion under shear conditions. Thus it is likely that elevated circulating levels of soluble CRP reduces the overall thrombotic potential.

The 80th anniversary of von Willebrand's disease: history, management and research

The history of von Willebrand's disease (VWD) is fascinating because it demonstrates how good clinical observations, genetic studies and biochemical skills can improve basic understanding of a disease and its management. The continuous efforts of scientists and clinicians during the last 80 years have significantly improved the knowledge of von Willebrand factor (VWF) structure and function and the management of VWD. Diagnosis of phenotype and genotype is now available in many countries and treatment is becoming more specific according to the VWD type. Any therapeutic agents must correct the dual defect of haemostasis, i.e. the abnormal platelet adhesion due to reduced and/or dysfunctional and low levels of factor VIII (FVIII) associated with VWF defects. Desmopressin (DDAVP) is the treatment of choice for type 1 VWD because it induces release of VWF from cellular compartments. Plasma virally inactivated VWF concentrates containing FVIII are effective and safe in patients unresponsive to DDAVP. There are advanced plans to develop a recombinant VWF but this product will require the concomitant administration of FVIII for the control of acute bleeds. Basic research studies on cellular biology, biochemistry and immunology have confirmed the role of VWF as a crucial participant in both haemostasis and thrombosis as its main biological activity is to support platelet adhesion-aggregation in the circulation. Retrospective and prospective clinical research studies, including bleeding history and laboratory markers for diagnosis as well as the use of DDAVP and VWF concentrates to manage or prevent bleeds in patients with VWD have been essential to provide general guidelines for VWD management. The large number of publications quoting VWD and VWF emphasizes the important role of VWF in medicine.

Localization and characterization of a novel secreted protein SCUBE1 in human platelets

The aim of the study was to investigate the protein expression and function of a novel secreted protein in the vascular system, named SCUBE1 for signal peptide, CUB (Complement proteins C1r/C1s, Uegf, and Bmp1) and epidermal growth factor-like (EGF)-like domain containing protein 1. Immunohistochemical analysis demonstrated that the SCUBE1 staining is mainly confined to the intravascular platelet-rich thrombus in vascular tissue samples. While quantitative real-time RT-PCR verified that the SCUBE1 mRNA is expressed in human platelets, numerous immunolocalization techniques revealed that the preformed SCUBE1 protein is stored in the alpha-granules and translocated to the surface upon platelet stimulation. A smaller SCUBE1 fragment, possibly formed by limited proteolysis after being released from the storage granules, was detected in thrombus lysate by Western blot analysis. Interestingly, deposition of SCUBE1 into the subendothelial matrix of the atherosclerotic plaques was evidenced by immunohistochemistry. In addition, studies of platelet adhesion and ristocetin-induced platelet agglutination showed that fragments containing the amino-terminal EGF-like repeats were able to support platelet adhesion and enhance the ristocetin-induced platelet agglutination, respectively. These data suggest that platelet-derived SCUBE1 could function as a novel adhesive molecule and its matrix-bound and soluble fragments may play critical (patho)physiological roles in cardiovascular biology.

Laminin stimulates spreading of platelets through integrin α6β1–dependent activation of GPVI

The extracellular matrix protein, laminin, supports platelet adhesion through binding to integrin alpha6beta1 In the present study, we demonstrate that human laminin, purified from placenta, also stimulates formation of filopodia and lamellipodia in human and mouse platelets through a pathway that is dependent on alpha6beta1 and the collagen receptor GPVI. The integrin alpha6beta1 is essential for adhesion to laminin, as demonstrated using an alpha6-blocking antibody, whereas GPVI is dispensable for this response, as shown using "knockout" mouse platelets. On the other hand, lamellipodia formation on laminin is completely inhibited in the absence of GPVI, although filopodia formation remains and is presumably mediated via alpha6beta1 Lamellipodia and filopodia formation are inhibited in Syk-deficient platelets, demonstrating a key role for the kinase in signaling downstream of GPVI and integrin alpha6beta1 GPVI was confirmed as a receptor for laminin using surface plasmon resonance spectroscopy and by demonstration of lamellipodia formation on laminin in the presence of collagenase. These results identify GPVI as a novel receptor for laminin and support a model in which integrin alpha6beta1 brings laminin to GPVI, which in turn mediates lamellipodia formation. We speculate that laminin contributes to platelet spreading in vivo through a direct interaction with GPVI.

The interaction of von Willebrand factor-A1 domain with collagen: mutation G1324S (type 2M von Willebrand disease) impairs the conformational change in A1 domain induced by collagen.

It is established that the A3 domain in von Willebrand factor (VWF) contains the major collagen-binding site. However, there are conflicting reports describing the capacity of the A1 domain to interact with collagen types I and III. In this study, we have used recombinant VWF-A1 polypeptides, as well as conformation-specific monoclonal antibodies (mAb), to analyze the A1-collagen interaction. The A1 domain bound to collagen with K(d) approximately 8.0 nm and this binding was blocked by the mAb 6G1, which blocks the interaction between ristocetin and VWF. In addition, collagen-bound A1 protein was able to support flow-dependent adhesion of platelets, demonstrating that the binding sites for collagen and glycoprotein (GP)Ib are different. Analysis with two conformation-specific mAb demonstrated that the structure of the A1 domain changed as a result of the binding to collagen. In contrast, the antibodies failed to detect conformational change in the G1324S mutant (type 2M von Willebrand disease). Thus, direct binding to collagen induces a change in the structural conformation within the VWF-A1 domain, and the G1324S substitution prevents this conformational change. This study has shown that the isolated A1 domain can simultaneously bind to collagen and platelet GPIb, supporting platelet adhesion under high-flow conditions. In addition, this study has used mAb to demonstrate that the binding of the isolated A1 domain or full-length VWF to collagen is accompanied by a conformational change in A1 domain.

Hepatology highlights

Hepatology highlights