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Abstract: Clot dissolution using enzymes from the human fibrinolytic system has long been a goal of physicians and scientists. While recent focus has been on activators of the fibrinolytic system, new data support the use of plasmin itself. This supplement reviews the role of plasmin in fibrinolysis and locally administered plasmin as a safe and effective therapeutic. [Copyright &y& Elsevier]

Kindlins play an important role in supporting integrin activation by cooperating with talin; however, the mechanistic details remain unclear. Here, we show that kindlins interacted directly with paxillin and that this interaction could support integrin αIIbβ3 activation. An exposed loop in the N-terminal F0 subdomain of kindlins was involved in mediating the interaction. Disruption of kindlin binding to paxillin by structure-based mutations significantly impaired the function of kindlins in supporting integrin αIIbβ3 activation. Both kindlin and talin were required for paxillin to enhance integrin activation. Interestingly, a direct interaction between paxillin and the talin head domain was also detectable. Mechanistically, paxillin, together with kindlin, was able to promote the binding of the talin head domain to integrin, suggesting that paxillin complexes with kindlin and talin to strengthen integrin activation. Specifically, we observed that crosstalk between kindlin-3 and the paxillin family in mouse platelets was involved in supporting integrin αIIbβ3 activation and in vivo platelet thrombus formation. Taken together, our findings uncover a novel mechanism by which kindlin supports integrin αIIbβ3 activation, which might be beneficial for developing safer anti-thrombotic therapies. [ABSTRACT FROM AUTHOR]

Immune complexes consisting of heparin, platelet factor 4 (PF4), and PF4/heparin-reactive antibodies are central to the pathogenesis of heparin-induced thrombocytopenia (HIT). It is as yet unclear what triggers the initial induction of pathogenic antibodies. We identified B cells in peripheral blood of healthy adults that produce PF4/heparin-specific antibodies following in vitro stimulation with proinflammatory molecules containing deoxycytosine-deoxyguanosine (CpG). Similarly, B cells from unmanipulated wild-type mice produced PF4/heparin-specific antibodies following in vitro or in vivo CpG stimulation. Thus, both healthy humans and mice possess preexisting inactive/tolerant PF4/heparin-specific B cells. The findings suggest that breakdown of tolerance leads to PF4/heparin-specific B-cell activation and antibody production in patients developing HIT. Consistent with this concept, mice lacking protein kinase Cδ (PKCδ) that are prone to breakdown of B-cell tolerance produced anti-PF4/heparin antibodies spontaneously. Therefore, breakdown of tolerance can lead to PF4/heparin-specific antibody production, and B-cell tolerance may play an important role in HIT pathogenesis. [ABSTRACT FROM AUTHOR]

Rap1b is activated by platelet agonists and plays a critical role in integrin αIIbβ3 inside-out signaling and platelet aggregation. Here we show that agonist-induced Rap1b activation plays an important role in stimulating secretion of platelet granules. We also show that αIIbβ3 outside-in signaling can activate Rap1b, and integrin outside-in signaling-mediated Rap1b activation is important in facilitating platelet spreading on fibrinogen and clot retraction. Rap1b-deficient platelets had diminished ATP secretion and P-selectin expression induced by thrombin or collagen. Importantly, addition of low doses of ADP and/or fibrinogen restored aggregation of Rap1b-deficient platelets. Furthermore, we found that Rap1b was activated by platelet spreading on immobilized fibrinogen, a process that was not affected by P2Y12 or TXA2 receptor deficiency, but was inhibited by the selective Src inhibitor PP2, the PKC inhibitor Ro-31-8220, or the calcium chelator demethyl-1,2-bis(2-aminophenoxy) ethane-N,N,Nβ,Nβ-tetraacetic acid tetrakis. Clot retraction was abolished, and platelet spreading on fibrinogen was diminished in Rap1b-deficient platelets compared with wild-type controls. The defects in clot retraction and spreading on fibrinogen of Rap1bdeficient platelets were not rescued by addition of MnCl2, which elicits αIIbβ3 outside-in signaling in the absence of inside-out signaling. Thus, our results reveal two different activation mechanisms of Rap 1 bas well as novel functions of Rap1 binplateletsecretion and in integrinαIIbβ3 outside-in signaling. [ABSTRACT FROM AUTHOR]

Rap1b, an abundant small GTPase in platelets, becomes rapidly activated upon stimulation with agonists. Though it has been implicated to act downstream from G protein-coupled receptors (GPCRs) and upstream of integrin alpha IIbbeta3, the precise role of Rap1b in platelet function has been elusive. Here we report the generation of a murine rap1b knockout and show that Rap1b deficiency results in a bleeding defect due to defective platelet function. Aggregation of Rap1b-null platelets is reduced in response to stimulation with both GPCR-linked and GPCR-independent agonists. Underlying the defective Rap1b-null platelet function is decreased activation of integrin alphaIIbbeta3 in response to stimulation with agonists and signaling downstream from the integrin alpha IIbbeta3. In vivo, Rap1b-null mice are protected from arterial thrombosis. These data provide genetic evidence that Rap1b is involved in a common pathway of integrin activation, is required for normal hemostasis in vivo, and may be a clinically relevant antithrombotic therapy target. [ABSTRACT FROM AUTHOR]

This study was undertaken to determine whether the response of smooth muscle cells to mitogens can be inhibited by inactivating ras with the ral GDS like protein-2 ras-binding domain (RGL2-RBD). RGL2 is a member of the ral GDS family of proteins that contains a carboxy terminal ras-binding domain which binds the GTP ligated form of ras and rap and a CDC25 homology domain with the structural features of a guanine nucleotide exchange factor. The effect of ras signaling on the smooth muscle cell growth factor response was studied using rat aortic A10 smooth muscle cells transfected with a plasmid that encoded the RGL2-RBD. RGL2-RBD transfection resulted in a 12-fold reduction in the number of clonal colonies that were obtained after selection, and dramatically slowed cell cycle progression. RGBL2-RBD reduced DNA synthesis and inhibited platelet derived growth factor (PDGF)-mediated activation of the MAPK pathway. These findings indicated that interfering with ras signaling inhibits smooth muscle cell proliferation and raise the possibility that ras signaling inhibition might be used therapeutically to control smooth muscle proliferation after vascular injury. [Copyright &y& Elsevier]

Mutations inLMAN1 (also calledERGIC-53) result in combined deficiency of factor V and factor VIII (F5F8D), an autosomal recessive bleeding disorder characterized by coordinate reduction of both clotting proteins[SUP1]. LMAN1 is a mannose-binding type 1 transmembrane protein localized to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC; refs. 2,3), suggesting that F5F8D could result from a defect in secretion of factor V and factor VIII (ref. 4). Correctly folded proteins destined for secretion are packaged in the ER into COPII-coated vesicles[SUP5], which subsequently fuse to form the ERGIC. Secretion of certain abundant proteins suggests a default pathway requiring no export signals (bulk flow; refs. 6,7). An alternative mechanism involves selective packaging of secreted proteins with the help of specific cargo receptors[SUP8-13]. The latter model would be consistent with mutations inLMAN1 causing a selective block to export of factor V and factor VIII. But - 30% of individuals with F5F8D have normal levels of LMAN1, suggesting that mutations in another gene may also be associated with F5F8D[SUP14,15]. Here we show that inactivating mutations in MCFD2 cause F5F8D with a phenotype indistinguishable from that caused by mutations in LMAN1. MCFD2 is localized to the ERGIC through a direct, calcium-dependent interaction with LMAN1. These findings suggest that the MCFD2-LMAN1 complex forms a specific cargo receptor for the ER-to-Golgi transport of selected proteins. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the effect of cross-linking and lyophilization on intracellular signalling processes in rehydrated, lyophilized (RL) platelets, which are under development as a platelet substitute for transfusion. Exposure of RL platelets to thrombin resulted in enhanced phosphorylation of several proteins, including 18 kDa and 42 kDa kinase substrates that were shown to be the substrates of myosin light chain and protein kinase C respectively. Cross-linking and lyophilization depleted the platelets of free cytoplasmic ADP and ATP, but had less effect on protein-bound nucleotides. The surface membrane of RL platelets was found to be permeable to poly dT probes less than approximately 3 kDa in size; larger nucleotide probes and proteins did not penetrate the surface membrane. Taken together, our results indicate that RL platelets retain some of the haemostatic stimulus-response functions of fresh platelets and are capable of feedback amplification in coagulation. [ABSTRACT FROM AUTHOR]

Background: Previously sharpin has been identified as an endogenous inhibitor of β1-integrin activation by directly binding to a conserved region in the cytoplasmic tails (CTs) of the integrin β1-associated α subunits. Methods: Here we employed biochemical approaches and cellular analyses to evaluate the function and molecular mechanism of the sharpin-kindlin-1 complex in regulating β1-integrin activation. Results: In this study, we found that although the inhibition of sharpin on β1-integrin activation could be confirmed, sharpin had no apparent effect on integrin αIIbβ3 activation in CHO cell system. Notably, a direct interaction between sharpin and the integrin β1 CT was detected, while the interaction of sharpin with the integrin αIIb and the β3 CTs were substantially weaker. Importantly, sharpin was able to inhibit the talin head domain binding to the integrin β1 CT, which can mechanistically contribute to inhibiting β1-integrin activation. Interestingly, we also found that sharpin interacted with kindlin-1, and the interaction between sharpin and the integrin β1 CT was significantly enhanced when kindlin-1 was present. Consistently, we observed that instead of acting as an activator, kindlin-1 actually suppressed the talin head domain mediated β1-integrin activation, indicating that kindlin-1 may facilitate recruitment of sharpin to the integrin β1 CT. Conclusion: Taken together, our findings suggest that sharpin may complex with both kindlin-1 and the integrin β1 CT to restrict the talin head domain binding, thus inhibiting β1-integrin activation. [ABSTRACT FROM AUTHOR]