Two-phase antithrombotic protection after anti-glycoprotein VI treatment in mice.

OBJECTIVE: Collagen and thrombin are the strongest physiological platelet agonists, acting through different receptors, among which glycoprotein VI (GPVI) and protease-activated receptors, respectively, are the essential ones. In mice, targeting of GPVI with the monoclonal antibody JAQ1 induces depletion of the receptor from circulating platelets, resulting in abolished collagen responses and long-lasting antithrombotic protection. METHODS AND RESULTS: Mice were treated with JAQ1, and the early effects of this treatment were analyzed. In addition to the known abolition of the collagen reactivity, this treatment also affected platelet response to thrombin but not other agonists. In platelets from JAQ1-treated mice, thrombin-induced activation of integrin alphaIIbbeta3, the surface expression of P-selectin, and the procoagulant activity were decreased on days 1 and 2, then progressively recovered and returned to normal on day 5. In parallel, the mice were transiently protected from lethal tissue factor-induced pulmonary thromboembolism (100% survivors versus 40% in control group), which appeared to be based on a decreased generation and activity of intravascular thrombin. CONCLUSIONS: Anti-GPVI treatment induces 2-phase antithrombotic protection in mice consisting of a partial and transient inhibition of thrombin responses in platelets and a prolonged and complete loss of the collagen response.

Relative antithrombotic effect of soluble GPVI dimer compared with anti-GPVI antibodies in mice.

Glycoprotein VI (GPVI) is an essential platelet collagen receptor; therefore, the inhibition of GPVI-collagen interactions may be an attractive antithrombotic strategy. We have previously shown that targeting of GPVI with antibodies leads to the depletion of the receptor and to long-term antithrombotic protection in mice. An alternative agent to interfere with GPVI-collagen interactions might be soluble GPVI acting as a competitive inhibitor, thereby averting undesired effects on platelets. To test this, we expressed soluble dimeric human GPVI, comprising the extracellular domain of the receptor fused to the human immunoglobulin Fc domain (GPVI-Fc), and compared its antithrombotic potential with that of anti-GPVI antibodies in mice. In contrast to a recent report, we found by intravital fluorescence microscopy and ultrasonic flow measurements that GPVI-Fc had no effect on platelet adhesion and thrombus formation at the injured arterial wall, whereas anti-GPVI antibodies profoundly inhibited these processes. Similar results were obtained with a fusion protein comprising the extracellular domain of mouse GPVI and human IgG-Fc. This indicates that direct targeting of GPVI provides significantly stronger protection against arterial thrombosis than soluble GPVI dimer.

Anti-glycoprotein VI treatment severely compromises hemostasis in mice with reduced alpha2beta1 levels or concomitant aspirin therapy.

BACKGROUND: Platelet inhibition is a major strategy to prevent arterial thrombosis, but it is frequently associated with increased bleeding because of impaired primary hemostasis. The activating platelet collagen receptor, glycoprotein VI (GP VI), may serve as a powerful antithrombotic target because its inhibition or absence results in profound protection against arterial thrombosis but no major bleeding in mice. METHODS AND RESULTS: Mice lacking (-/-) or expressing half-levels (+/-) of the other major platelet collagen receptor, integrin alpha2beta1, were injected with the anti-GP VI antibody JAQ1 and analyzed on day 5. Anti-GP VI treatment resulted in a marked hemostatic defect in alpha2-/- or alpha2+/- mice, as shown by dramatically prolonged tail bleeding times. Platelet adhesion to collagen was studied in an ex vivo whole-blood perfusion system under high shear conditions. Weak integrin activation by thromboxane A2 (TxA2) receptor stimulation restored defective adhesion of anti-GP VI-treated wild-type but not alpha2-/- or alpha2+/- platelets to collagen. This process required the simultaneous activation of the G(q) and G13 signaling pathways, as demonstrated by use of the respective knockout strains. Conversely, inhibition of TxA2 production by aspirin severely compromised hemostasis in anti-GP VI-treated or GP VI/Fc receptor gamma-chain-deficient but not control mice. CONCLUSIONS: Anti-GP VI therapy may result in defective hemostasis in patients with reduced alpha2beta1 levels or concomitant aspirin therapy. These observations may have important implications for a potential use of anti-GP VI-based therapeutics in the prevention of cardiovascular disease.

Facilitating roles of murine platelet glycoprotein Ib and alphaIIbbeta3 in phosphatidylserine exposure during vWF-collagen-induced thrombus formation.

Vessel wall damage exposes collagen fibres, to which platelets adhere directly via the collagen receptors glycoprotein (GP) VI and integrin alpha(2)beta(1) and indirectly by collagen-bound von Willebrand factor (vWF) via the GPIb-V-IX and integrin alphaIIbbeta3 receptor complexes. Platelet-collagen interaction under shear stimulates thrombus formation in two ways, by integrin-dependent formation of platelet aggregates and by surface exposure of procoagulant phosphatidylserine (PS). GPVI is involved in both processes, complemented by alpha2beta1. In mouse blood flowing over collagen, we investigated the additional role of platelet-vWF binding via GPIb and alphaIIbbeta3. Inhibition of GPIb as well as blocking of vWF binding to collagen reduced stable platelet adhesion at high shear rate. This was accompanied by delayed platelet Ca(2+) responses and reduced PS exposure, while microaggregates were still formed. Inhibition of integrin alphaIIbbeta3 with JON/A antibody, which blocks alphaIIbbeta3 binding to both vWF and fibrinogen, reduced PS exposure and aggregate formation. The JON/A effects were not enhanced by combined blocking of GPIb-vWF binding, suggesting a function for alphaIIbbeta3 downstream of GPIb. Typically, with blood from FcR gamma-chain +/- mutant mice, expressing 50% of normal platelet GPVI levels, GPIb blockage almost completely abolished platelet adhesion and PS exposure. Together, these data indicate that, under physiological conditions of flow, both adhesive receptors GPIb and alphaIIbbeta3 facilitate GPVI-mediated PS exposure by stabilizing platelet binding to collagen. Hence, these glycoproteins have an assistant procoagulant role in collagen-dependent thrombus formation, which is most prominent at reduced GPVI activity and is independent of the presence of thrombin.

Needle tip control and its effect in reducing intraoperative complications during tension-free vaginal tape placement.

Tactile needle tip control was used to aid perforation during standard tension-free vaginal tape (TVT) placement to treat urinary incontinence. The success and complications of this novel method were compared retrospectively with the reported results of the standard technique. One hundred nine patients had TVT placement between 1998 and 2001, with follow-up continuing into 2002. Preoperatively, the severity of urinary incontinence was assessed objectively. Postoperatively, TVT effectiveness was assessed subjectively by standardized questionnaire, completed by 78 of the 109 patients (72%). Objective 2-year rates for continence or improvement and most complication rates were similar to previously reported results. Needle tip control was helpful in lowering the occurrence of bladder perforation. Longer postoperative recovery times associated with postoperative dysuria or obstruction did not decrease patient satisfaction if the patient experienced a marked improvement in leakage.

G13 is an essential mediator of platelet activation in hemostasis and thrombosis.

Platelet activation at sites of vascular injury is essential for primary hemostasis, but also underlies arterial thrombosis leading to myocardial infarction or stroke. Platelet activators such as adenosine diphosphate, thrombin or thromboxane A(2) (TXA(2)) activate receptors that are coupled to heterotrimeric G proteins. Activation of platelets through these receptors involves signaling through G(q), G(i) and G(z) (refs. 4-6). However, the role and relative importance of G(12) and G(13), which are activated by various platelet stimuli, are unclear. Here we show that lack of Galpha(13), but not Galpha(12), severely reduced the potency of thrombin, TXA(2) and collagen to induce platelet shape changes and aggregation in vitro. These defects were accompanied by reduced activation of RhoA and inability to form stable platelet thrombi under high shear stress ex vivo. Galpha(13) deficiency in platelets resulted in a severe defect in primary hemostasis and complete protection against arterial thrombosis in vivo. We conclude that G(13)-mediated signaling processes are required for normal hemostasis and thrombosis and may serve as a new target for antiplatelet drugs.

Multiple integrin-ligand interactions synergize in shear-resistant platelet adhesion at sites of arterial injury in vivo.

Damage to the integrity of the vessel wall results in exposure of the subendothelial extracellular matrix (ECM), which triggers integrin-dependent adhesion and aggregation of platelets. The role of platelet beta1 integrins in these processes remains mostly undefined. Here, we demonstrate by intravital fluorescence microscopy that platelet adhesion and thrombus growth on the exposed ECM of the injured carotid artery is not significantly altered in alpha2-null mice and even in mice with a Cre/loxP-mediated loss of all beta1 integrins on their platelets. In contrast, inhibition of alphaIIbbeta3 integrin on platelets in wild-type mice blocked aggregate formation and reduced platelet adhesion by 60.0%. Strikingly, alphaIIbbeta3 inhibition had a comparable effect in alpha2-null mice, demonstrating that other receptors mediate shear-resistant adhesion in the absence of functional alpha2beta1 and alphaIIbbeta3. These were identified to be alpha5beta1 and/or alpha6beta1 as alphaIIbbeta3 inhibition abrogated platelet adhesion in beta1-null mice. We conclude that shear-resistant platelet adhesion on the injured vessel wall in vivo is a highly integrated process involving multiple integrin-ligand interactions, none of which by itself is essential.

Transvaginal tape lysis for urinary obstruction after tension-free vaginal tape placement.

PURPOSE: We examine the outcome of tape lysis in subjects with obstructive urinary retention after initial tension-free vaginal tape placement. MATERIALS AND METHODS: Charts from 109 consecutive subjects who underwent tension-free vaginal tape placement from 1998 to 2001 were retrospectively reviewed. Of the subjects 78 (72%) consented to participation and completion of a questionnaire. All subjects underwent a routine preoperative evaluation before surgery and followup was implemented through 2002. Tape lysis using a standard technique was performed in 5 patients with documented obstructive urinary retention. Subjective assessment by the validated Bristol Lower Urinary Tract Symptoms questionnaire and a postoperative survey characterized perceptions of incontinence, results of initial tension-free vaginal tape placement and outcome of subsequent tape lysis. RESULTS: The objective incontinence cure rate was 89%. Of the 78 patients 17 (22%) and 21 (27%) experienced transience in either dysuria or incomplete bladder emptying, respectively. Transection for obstructive urinary retention was successfully accomplished in 5 subjects with 4 (80%) remaining continent. CONCLUSIONS: Tape lysis is simple and effective for relieving obstruction, and is not associated with a high rate of recurrent stress urinary incontinence.

Targeting of platelet integrin alphaIIbbeta3 determines systemic reaction and bleeding in murine thrombocytopenia regulated by activating and inhibitory FcgammaR.

Previous work on cellular destruction induced by several clinically relevant anti-platelet IgG antibodies suggested antigen-specific mechanisms in the development of immune thrombocytopenia in mice. mAb directed against mouse platelet GPIbalpha and integrin alpha(IIb)beta(3) were highly pathogenic, and mediated their effects via different Fc-dependent (alpha(IIb)beta(3)) and Fc-independent (GPIbalpha) pathways, indicating that clearance of IgG-bound platelets is only one event in the pathogenesis of murine thrombocytopenia. Here, we demonstrate that in addition to thrombocytopenia, targeting of platelet integrin alpha(IIb)beta(3) results in acute systemic reaction and bleeding that is regulated by activating IgG Fc receptors (FcgammaR) and the inhibitory FcgammaRII. As shown by electron microscopy, anti-alpha(IIb)beta(3) IgG mediated initial loss of alpha(IIb)beta(3) integrin from platelet surfaces followed by rapid accumulation of alpha(IIb)beta(3) antibody-containing immune complex (IC)-like structures in spleen and liver in vivo. In FcRgamma chain deficiency, mice resisted bleeding, but not platelet destruction, while genetic ablation of FcgammaRII resulted in uncontrolled systemic reaction and severe hemorrhage leading to enhanced mortality. Together, these results provide evidence that IC formation and engagement of FcgammaR on effector cells determines the alpha(IIb)beta(3)-specific part of the platelet pathology of the systemic reaction and bleeding in murine thrombocytopenia.

Complementary roles of glycoprotein VI and alpha2beta1 integrin in collagen-induced thrombus formation in flowing whole blood ex vivo.

Platelets interact vigorously with subendothelial collagens that are exposed by injury or pathological damage of a vessel wall. The collagen-bound platelets trap other platelets to form aggregates, and they expose phosphatidylserine (PS) required for coagulation. Both processes are implicated in the formation of vaso-occlusive thrombi. We previously demonstrated that the immunoglobulin receptor glycoprotein VI (GPVI), but not integrin alpha2beta1, is essential in priming platelet-collagen interaction and subsequent aggregation. Here, we report that these receptors have yet a complementary function in ex vivo thrombus formation during perfusion of whole blood over collagen. With mice deficient in GPVI or blocking antibodies, we found that GPVI was indispensable for collagen-dependent Ca2+ mobilization, exposure of PS, and aggregation of platelets. Deficiency of integrin beta1 reduces the GPVI-evoked responses but still allows the formation of loose platelet aggregates. By using mice deficient in G(alpha)q or specific thromboxane A2 and ADP antagonists, we show that these autocrine agents mediated aggregation but not collagen-induced Ca2+ mobilization or PS exposure. Collectively, these data indicate that integrin alpha2beta1 facilitates the central function of GPVI in the platelet activation processes that lead to thrombus formation, whereas the autocrine thromboxane A2 and ADP serve mainly to trigger aggregate formation.

Targeting of the collagen-binding site on glycoprotein VI is not essential for in vivo depletion of the receptor.

Glycoprotein (GP) VI is an essential collagen receptor on platelets and may serve as an attractive target for antithrombotic therapy. We have previously shown that a monoclonal antibody (mAb) against the major collagen-binding site on mouse GPVI (JAQ1) induces irreversible down-regulation of the receptor and, consequently, long-term antithrombotic protection in vivo. To determine whether this unique in vivo effect of JAQ1 is based on its interaction with the ligand-binding site on GPVI, we generated new mAbs against different epitopes on GPVI (JAQ2, JAQ3) and tested their in vitro and in vivo activity. We show that none of the mAbs inhibited platelet activation by collagen or the collagen-related peptide in vitro. Unexpectedly, however, injection of either antibody induced depletion of GPVI with the same efficacy and kinetics as JAQ1. Importantly, this effect was also seen with monovalent F(ab) fragments of JAQ2 and JAQ3, excluding the involvement of the Fc part or the dimeric form of anti-GPVI antibodies in this process. This indicates that anti-GPVI agents, irrespective of their binding site may generally induce down-regulation of the receptor in vivo.

A crucial role of glycoprotein VI for platelet recruitment to the injured arterial wall in vivo.

Platelet adhesion and aggregation at sites of vascular injury is crucial for hemostasis but may lead to arterial occlusion in the setting of atherosclerosis and precipitate diseases such as myocardial infarction. A current hypothesis suggests that platelet glycoprotein (GP) Ib interaction with von Willebrand factor recruits flowing platelets to the injured vessel wall, where subendothelial fibrillar collagens support their firm adhesion and activation. However, so far this hypothesis has not been tested in vivo. Here, we demonstrate by intravital fluorescence microscopy of the mouse carotid artery that inhibition or absence of the major platelet collagen receptor, GPVI, abolishes platelet-vessel wall interactions after endothelial denudation. Unexpectedly, inhibition of GPVI by the monoclonal antibody JAQ1 reduced platelet tethering to the subendothelium by approximately 89%. In addition, stable arrest and aggregation of platelets was virtually abolished under these conditions. Using different models of arterial injury, the strict requirement for GPVI in these processes was confirmed in GPVI-deficient mice, where platelets also failed to adhere and aggregate on the damaged vessel wall. These findings reveal an unexpected role of GPVI in the initiation of platelet attachment at sites of vascular injury and unequivocally identify platelet-collagen interactions (via GPVI) as the major determinant of arterial thrombus formation.

Costimulation of Gi- and G12/G13-mediated signaling pathways induces integrin alpha IIbbeta 3 activation in platelets.

Platelet activation is a complex process induced by a variety of stimuli, which act in concert to ensure the rapid formation of a platelet plug at places of vascular injury. We show here that fibrillar collagen, which initiates platelet activation at the damaged vessel wall, activates only a small fraction of platelets in suspension directly, whereas the majority of platelets becomes activated by mediators released from collagen-activated platelets. In Galpha(q)-deficient platelets that do not respond with activation of integrin alpha(IIb)beta(3) to a variety of mediators like thromboxane A2 (TXA2), thrombin, or ADP, collagen at high concentrations was able to induce aggregation, an effect that could be blocked by antagonists of the TXA2 or P2Y12 receptors. The activation of TXA2 or P2Y12 receptors alone, which in Galpha(q)-deficient platelets couple to G12/G13 and Gi, respectively, did not induce platelet integrin activation or aggregation. However, concomitant activation of both receptors resulted in irreversible integrin alpha(IIb)beta3-mediated aggregation of Galpha(q)-deficient platelets. Thus, the activation of G12/G13- and Gi-mediated signaling pathways is sufficient to induce integrin alpha(IIb)beta3 activation. Although G(q)-mediated signaling plays an important role in platelet activation, it is not strictly required for the activation of integrin alpha(IIb)beta3. This indicates that the efficient induction of platelet aggregation through G-protein-coupled receptors is an integrated response mediated by various converging G-protein-mediated signaling pathways involving G(q) and G(i) as well as G12/G13.

Flow cytometric detection of activated mouse integrin alphaIIbbeta3 with a novel monoclonal antibody.

BACKGROUND: Integrin alphaIIbbeta3 mediates platelet adhesion and aggregation and plays a crucial role in thrombosis and hemostasis. alphaIIbbeta3 is expressed in a low affinity state on resting platelets. Upon platelet activation, alphaIIbbeta3 shifts to a high affinity conformation that efficiently binds its ligands. On human platelets, the high affinity conformation of alphaIIbbeta3 is detected by the monoclonal antibody (mAb), PAC-1. However, a reagent with binding specificity to high affinity mouse alphaIIbbeta3 has not been described so far. METHODS: A novel rat mAb directed against mouse alphaIIbbeta3 (JON/A) was generated and characterized. JON/A was conjugated with fluorescein isothiocyanate (JON/A(FITC)) or with R-phycoerythrin (JON/A(PE)) and used for flow cytometric analysis of mouse platelets. RESULTS: Although JON/A(FITC) bound to resting and activated platelets, virtually no binding of the larger JON/A(PE) to resting platelets was detectable. However, strong binding of JON/A(PE) occurred on platelet activation in a dose-dependent manner. Binding of JON/A(PE) required extracellular free calcium and was irreversible, thereby stabilizing the high affinity conformation of alphaIIbbeta3. CONCLUSION: JON/A(PE) is the first tool for direct assessment of integrin alphaIIbbeta3 activation in mice. Furthermore, JON/A(FITC) and JON/A(PE) provide the first examples of fluorescent antibody derivatives with identical antigenic specificity that allow the discrimination between the resting and the activated state of an integrin.

Differential effects of reduced glycoprotein VI levels on activation of murine platelets by glycoprotein VI ligands.

We have investigated the effects of decreased levels of the complex between glycoprotein VI (GPVI) and the Fc receptor gamma-chain (FcRgamma) on responses to collagen and GPVI-specific ligands in murine platelets. We show that levels of GPVI-FcRgamma of the order of 50% and 20% of wild-type levels caused 2- and 5-fold shifts to the right respectively in the dose-response curve for aggregation in response to collagen, the snake toxin convulxin and the monoclonal antibody JAQ1. In addition, there is a delay in the onset of aggregation in response to collagen. In contrast, the stimulation of protein tyrosine phosphorylation by collagen (as measured after 150 s) and adhesion to a collagen-coated surface under static conditions were unaffected in platelets with 50% and 20% of wild-type levels of GPVI. In contrast, responses to a collagen-related peptide (CRP), made up of repeat glycine-proline-hydroxyproline motifs, were markedly inhibited and abolished in platelets expressing 50% and 20% of wild-type levels of GPVI respectively. We suggest that the marked effect of a reduction in GPVI levels on the CRP-induced activation of platelets is due to the multivalent nature of CRP and the fact that GPVI is its sole receptor on platelets. Thus it appears that the interaction of CRP with GPVI is determined by a combination of affinity and avidity. The observation that collagen does not behave like CRP in platelets expressing reduced levels of GPVI, even in the combined presence of blocking antibodies against integrin alpha2beta1 and GPV, suggests that collagen has a greater affinity than CRP for GPVI, and/or that other receptors are involved in its binding to platelets. The clinical significance of these results is discussed.

Integrin alpha 2-deficient mice develop normally, are fertile, but display partially defective platelet interaction with collagen.

The integrin alpha(2)-subunit was ablated in mice by targeted deletion of the ITGA2 gene. alpha(2)-Deficient animals develop normally, are fertile, and reproduce. Surprisingly, no obvious anatomical or histological differences were observed in mutant mice. Besides its significance in tissue morphogenesis, integrin alpha(2)beta(1) has been reported to play a major role in hemostasis by mediating platelet adhesion and activation on subendothelial collagen. To define its role in hemostasis, alpha(2)-deficient platelets were analyzed for their capacity to adhere to and aggregate in response to fibrillar or soluble collagen type I. We show that aggregation of alpha(2)-deficient platelets to fibrillar collagen is delayed but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, alpha(2)-deficient platelets normally adhere to fibrillar collagen. However, in the presence of an antibody against GPVI (activating platelet collagen receptor), adhesion of alpha(2)-deficient but not wild type platelets is abrogated. These results demonstrate that integrin alpha(2)beta(1) significantly contributes to platelet adhesion to (fibrillar) collagen, which is further confirmed by the abolished adhesion of alpha(2)-deficient platelets to soluble collagen. Thus, alpha(2)beta(1) plays a supportive rather than an essential role in platelet-collagen interactions. These results are in agreement with the observation that alpha(2)beta(1)-deficient animals suffer no bleeding anomalies.