C.Brakebusch and W.Bergmeier contributed equally to this workPlatelet adhesion on and activation by components of the extracellular matrix are crucial to arrest posttraumatic bleeding, but can also harm tissue by occluding diseased vessels. Integrin a2b1 is thought to be essential for platelet adhesion to subendothelial collagens, facilitating subsequent interactions with the activating platelet collagen receptor, glycoprotein VI (GPVI). Here we show that Cre/loxP-mediated loss of b1 integrin on platelets has no signi®cant effect on the bleeding time in mice. Aggregation of b1-null platelets to native ®brillar collagen is delayed, but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, b1-null platelets adhere to ®brillar, but not soluble collagen under static as well as low (150 s ±1 ) and high (1000 s ±1 ) shear ow conditions, probably through binding of aIIbb3 to von Willebrand factor. On the other hand, we show that platelets lacking GPVI can not activate integrins and consequently fail to adhere to and aggregate on ®brillar as well as soluble collagen. These data show that GPVI plays the central role in platelet±collagen interactions by activating different adhesive receptors, including a2b1 integrin, which strengthens adhesion without being essential. Keywords: collagen/Cre/loxP/GPVI/a2b1 integrin/ platelets IntroductionDamage to the integrity of the vessel wall results in exposure of the subendothelial extracellular matrix (ECM), which triggers adhesion and aggregation of platelets (Weiss, 1975). The consequence of this process is the formation of a thrombus, which prevents blood loss at sites of injury or leads to occlusion and irreversible tissue damage or infarction in diseased vessels. Integrins play a central role in adhesion and aggregation of platelets (Phillips et al., 1991;Shattil et al., 1998). Integrins are heterodimeric transmembrane receptors composed of an a and a b subunit. Resting platelets express their integrins in a low af®nity state. After activation, mediated by other platelet receptors, integrins shift to a high af®nity state and bind their ligands ef®ciently (Phillips et al., 1991;Shattil et al., 1998).The ECM of the vessel walls is rich in collagens, which play essential roles in thrombus formation by providing a substrate for platelet adhesion and by activating platelets (Baumgartner, 1977). Besides GPIb-V-IX and aIIbb3 integrin, which interact indirectly with collagen via von Willebrand factor (vWF) (Savage et al., 1998), a large number of collagen receptors have been identi®ed on platelets, including most importantly a2b1 integrin (Santoro, 1986) and glycoprotein VI (GPVI) (Moroi et al., 1989). A current model of platelet adhesion to collagen suggests that the GPIb±vWF interaction mediates initial tethering of platelets at high shear, followed by a2b1 integrin-mediated ®rm adhesion, which halts platelet translocation and allows collagen interactions with GPVI, ®nally resulting in platelet activation and thrombus growth (Sixma et...

Coronary artery thrombosis is often initiated by abrupt disruption of the atherosclerotic plaque and activation of platelets on the subendothelial layers in the disrupted plaque. The extracellular matrix protein collagen is the most thrombogenic constituent of the subendothelial layer; therefore, a selective inhibition of the collagen activation pathway in platelets may provide strong antithrombotic protection while preserving other platelet functions. Here we demonstrate that treatment of mice with a monoclonal antibody against the activating platelet collagen receptor glycoprotein VI (GPVI; JAQ1) results in specific depletion of the receptor from circulating platelets and abolished responses of these cells to collagen and collagen-related peptides (CRPs). JAQ1-treated mice were completely protected for at least 2 wk against lethal thromboembolism induced by infusion of a mixture of collagen (0.8 mg/kg) and epinephrine (60 μg/ml). The tail bleeding times in JAQ1-treated mice were only moderately increased compared with control mice probably because the treatment did not affect platelet activation by other agonists such as adenosine diphosphate or phorbol myristate acetate. These results suggest that GPVI might become a target for long-term prophylaxis of ischemic cardiovascular diseases and provide the first evidence that it is possible to specifically deplete an activating glycoprotein receptor from circulating platelets in vivo.

Peri-operative SARS-CoV-2 infection increases postoperative mortality. The aim of this study was to determine the optimal duration of planned delay before surgery in patients who have had SARS-CoV-2 infection. This international, multicentre, prospective cohort study included patients undergoing elective or emergency surgery during October 2020. Surgical patients with pre-operative SARS-CoV-2 infection were compared with those without previous SARS-CoV-2 infection. The primary outcome measure was 30-day postoperative mortality. Logistic regression models were used to calculate adjusted 30-day mortality rates stratified by time from diagnosis of SARS-CoV-2 infection to surgery. Among 140,231 patients (116 countries), 3127 patients (2.2%) had a pre-operative SARS-CoV-2 diagnosis. Adjusted 30-day mortality in patients without SARS-CoV-2 infection was 1.5% (95%CI 1.4-1.5). In patients with a pre-operative SARS-CoV-2 diagnosis, mortality was increased in patients having surgery within 0-2 weeks, 3-4 weeks and 5-6 weeks of the diagnosis (odds ratio (95%CI) 4.1 (3.3-4.8), 3.9 (2.6-5.1) and 3.6 (2.0-5.2), respectively). Surgery performed ≥ 7 weeks after SARS-CoV-2 diagnosis was associated with a similar mortality risk to baseline (odds ratio (95%CI) 1.5 (0.9-2.1)). After a ≥ 7 week delay in undertaking surgery following SARS-CoV-2 infection, patients with ongoing symptoms had a higher mortality than patients whose symptoms had resolved or who had been asymptomatic (6.0% (95%CI 3.2-8.7) vs. 2.4% (95%CI 1.4-3.4) vs. 1.3% (95%CI 0.6-2.0), respectively). Where possible, surgery should be delayed for at least 7 weeks following SARS-CoV-2 infection. Patients with ongoing symptoms ≥ 7 weeks from diagnosis may benefit from further delay.

Platelet glycoprotein (GP) VI has been proposed as the major collagen receptor for activation of human platelets. Human GPVI belongs to the immunoglobulin superfamily and is noncovalently associated with the FcR␥ chain that is involved in signaling through the receptor. In mice, similar mechanisms seem to exist as platelets from FcR␥ chain-deficient mice do not aggregate in response to collagen. However, the activating collagen receptor on mouse platelets has not been definitively identified. In the current study we examined the function and in vivo expression of GPVI in control and FcR␥ chain-deficient mice with the first monoclonal antibody against GPVI (JAQ1). On wild type platelets, JAQ1 inhibited platelet aggregation induced by collagen but not PMA or thrombin. Cross-linking of bound JAQ1, on the other hand, induced aggregation of wild type but not FcR␥ chain-deficient platelets. JAQ1 stained platelets and megakaryocytes from wild type but not FcR␥ chaindeficient mice. Furthermore, JAQ1 recognized GPVI (approximately 60 kDa) in immunoprecipitation and Western blot experiments with wild type but not FcR␥ chain-deficient platelets. These results strongly suggest that GPVI is the collagen receptor responsible for platelet activation in mice and demonstrate that the association with the FcR␥ chain is critical for its expression and function.Collagen is one of the major components of the vessel wall responsible for platelet adhesion and activation at sites of vascular injury (1). A variety of collagens have been identified, seven of which are found in the subendothelial layer. The interaction between platelets and collagen can either occur indirectly via intermediary proteins like von Willebrand factor, which complexes to collagen(s) in the vessel wall and concomitantly binds to the platelet receptors glycoprotein (GP) 1 Ib-

The pathogenic effects of antiplatelet antibodies were investigated in mice. Monoclonal antibodies (mAbs) of different immunoglobulin G subclass directed against mouse GPIIbIIIa, GPIIIa, GPIbα, GPIb-IX, GPV, and CD31 were generated and characterized biochemically. MAbs against GPIb-IX, GPV, CD31, and linear epitopes on GPIIIa had mild and transient effects on platelet counts and induced no spontaneous bleeding. Anti-GPIbα mAbs induced profound irreversible thrombocytopenia (< 3% of normal) by Fc-independent mechanisms but only had minor effects on hematocrits. In contrast, injection of intact mAbs, but not F(ab)2 fragments, against conformational epitopes on GPIIbIIIa, induced irreversible thrombocytopenia, acute systemic reactions, hypothermia, decreased hematocrits, and a paradoxical loss of surface GPIIbIIIa on platelets in vivo, the latter suggesting the formation of platelet-derived microparticles. Blockage of platelet-activating factor receptors inhibited the acute reactions, but not thrombocytopenia, loss of GPIIbIIIa, and decreases in hematocrits. Repeated injections of low doses of anti-GPIIbIIIa antibodies resulted in profound thrombocytopenia and bleeding, whereas no acute systemic reactions were observed. These data strongly suggest that the identity of the target antigen recognized by antiplatelet antibodies determines the mechanisms of platelet destruction and the severity of bleeding in mice, the latter depending on previously unrecognized anti-GPIIbIIIa-specific inflammatory mechanisms.