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Circulating platelets contain high concentrations of TGF-β1 in their α-granules and release it on platelet adhesion/activation. We hypothesized that uncontrolled in vitro release of platelet TGF-β1 may confound measurement of plasma TGF-β1 in mice and that in vivo release and activation may contribute to cardiac pathology in response to constriction of the transverse aorta, which produces both high shear and cardiac pressure overload. Plasma TGF-β1 levels in blood collected from C57Bl/6 mice by the standard retro-bulbar technique were much higher than those obtained when prostaglandin E₁ was added to inhibit release or when blood was collected percutaneously from the left ventricle under ultrasound guidance. Even with optimal blood drawing, plasma TGF-β1 was lower in mice rendered profoundly thrombocytopenic or mice with selectively low levels of platelet TGF-β1 because of megakaryocyte-specific disruption of their TGF-β1 gene (Tgfb1(flox)). Tgfb1(flox) mice were also partially protected from developing cardiac hypertrophy, fibrosis, and systolic dysfunction in response to transverse aortic constriction. These studies demonstrate that plasma TGF-β1 levels can be assessed accurately, but it requires special precautions; that platelet TGF-β1 contributes to plasma levels of TGF-β1; and that platelet TGF-β1 contributes to the pathologic cardiac changes that occur in response to aortic constriction.

Background and Objective: Platelets are essential for hemostasis, and they cause resistance to fibrinolysis by tissue-type plasminogen activator. In contrast, platelets enhance fibrinolysis mediated by single-chain urokinase-type plasminogen activator (scu-PA). This study investigated the mechanism behind this profibrinolytic role of platelets., Methods and Results: Platelets enhanced scu-PA activity, but not urokinase-type plasminogen activator (u-PA) activity, in plasma clot lysis and chromogenic assays. We established, using the non-cleavable scu-PA mutant (Lys158-->Glu) and protease inhibitors, that platelets increased activation to u-PA by a serine protease. Activation of scu-PA was platelet-dependent, even in plasma. It occurred in platelet-rich but not in platelet-poor plasma, as assessed by sodium dodecylsulfate polyacrylamide gel electrophoresis and zymography after addition of plasminogen activator inhibitor-1. Candidate proteases that are known to activate scu-PA and are present in platelet preparations were investigated. Factor VII activating protease was detected in platelet preparations by western blotting, but its inhibition by antibodies did not inhibit activation of scu-PA by platelets. Plasmin and plasma kallikrein both mimicked the platelet effect, but were distinguished by their responses to a range of inhibitors. Analysis of platelet-associated protease activity and the time course of scu-PA activation pointed towards plasminogen, and the data were consistent with a mechanism of reciprocal activation. The essential role of plasminogen was revealed using platelets from plasminogen-deficient mice, which could not activate scu-PA. Local plasminogen on platelet membranes was markedly more effective than solution-phase plasminogen in activation of scu-PA., Conclusions: Platelets enhance fibrinolysis by scu-PA through reciprocal activation of scu-PA and platelet-associated plasminogen, a system that is potentially important in the lysis of platelet-rich thrombi.

Platelet P-selectin plays important roles in inflammation and contributes to thrombosis and hemostasis. Although it has been reported that von Willebrand factor (VWF) affects P-selectin expression on endothelial cells, little information is available regarding regulation of platelet P-selectin expression. Here, we first observed that P-selectin expression was significantly decreased on platelets of fibrinogen and VWF double-deficient mice. Subsequently, we identified this was due to fibrinogen deficiency. Impaired P-selectin expression on fibrinogen-deficient platelets was further confirmed in human hypofibrinogenemic patients. We demonstrated that this impairment is unlikely due to excessive P-selectin shedding, deficient fibrinogen-mediated cell surface P-selectin binding, or impaired platelet granule release, but rather is due to decreased platelet P-selectin content. Fibrinogen transfusion completely recovered this impairment in fibrinogen-deficient (Fg(-/-)) mice, and engagement of the C-terminus of the fibrinogen gamma chain with beta3 integrin was required for this process. Furthermore, Fg(-/-) platelets significantly increased P-selectin expression following transfusion into beta3 integrin-deficient mice and when cultured with fibrinogen. These data suggest fibrinogen may play important roles in inflammation, thrombosis, and hemostasis via enhancement of platelet P-selectin expression. Since human fibrinogen levels vary significantly in normal and diseased populations, P-selectin as an activation marker on platelets should be used with caution.

We investigated the assembly of soluble fibronectin by lysophosphatidic acid-activated platelets adherent to fibrinogen or fibrin. More fibronectin was assembled by activated platelets spread on fibrin matrices than by platelets spread on adsorbed fibrinogen. The difference between platelets adherent to fibrinogen and fibrin occurred under both static and flow conditions. Similar differences were seen in binding of the 70-kDa N-terminal fragment of fibronectin that recognizes fibronectin assembly sites on adherent cells. Antibody and peptide blocking studies demonstrated that alphaIIb beta3 integrin mediates platelet adhesion to fibrinogen, whereas both alphav beta3 and alphaIIb beta3 mediate platelet adhesion to fibrin. The hypothesis that engagement of the C-terminal QAGDV sequence of the fibrinogen gamma-chain by alphaIIb beta3 inhibits the ability of the platelet to assemble fibronectin was tested by several experiments. Activated platelets adherent to adsorbed mutant fibrinogen lacking the QAGDV sequence (gammadelta5FG) were assembly-competent, as were platelets adherent to adsorbed normal fibrinogen that had been pretreated with the 7E9 antibody to the C terminus of the gamma-chain. Moreover, adsorbed normal fibrinogen but not gammadelta5FG suppressed the ability of co-adsorbed fibronectin to direct assembly of soluble fibronectin by spread platelets. The suppressive effect was lost when a surface of co-adsorbed fibronectin and fibrinogen was pretreated with 7E9. These results support a model in which the engagement of alphaIIb beta3 by the C-terminal sequence of the fibrinogen gamma-chain initiates signals that suppress subsequent fibronectin assembly by spread platelets. This interaction is less dominant when platelets adhere to fibrin, resulting in enhanced fibronectin assembly.

To test the hypothesis that platelet activation contributes to tumor dissemination, we studied metastasis in mice lacking Galphaq, a G protein critical for platelet activation. Loss of platelet activation resulted in a profound diminution in both experimental and spontaneous metastases. Analyses of the distribution of radiolabeled tumor cells demonstrated that platelet function, like fibrinogen, significantly improved the survival of circulating tumor cells in the pulmonary vasculature. More detailed studies showed that the increase in metastatic success conferred by either platelets or fibrinogen was linked to natural killer cell function. Specifically, the pronounced reduction in tumor cell survival observed in fibrinogen- and Galphaq-deficient mice relative to control animals was eliminated by the immunologic or genetic depletion of natural killer cells. These studies establish an important link between hemostatic factors and innate immunity and indicate that one mechanism by which the platelet-fibrin(ogen) axis contributes to metastatic potential is by impeding natural killer cell elimination of tumor cells.

Studies of mice with genetic deficits in specific coagulation factors have shown that many, but not all, components of the hemostatic system serve an essential role in mouse embryogenesis and pregnancy. Although the developmental failures observed in these mice are typically associated with severe hemorrhage, it is uncertain whether the role of coagulation factors in embryogenesis and reproduction is specifically tied to their function in thrombus formation and prevention of blood loss (i.e. hemostasis). Here, we show (i) that a complete loss of fibrinogen- and platelet-dependent hemostatic capacity does not reproduce the developmental defects occurring in mice with either deficits in thrombin generation or unfettered thrombin consumption; (ii) that the essential role of fibrinogen in the maintenance of pregnancy does not involve interaction with platelets; and (iii) that the previously described in utero growth retardation of gene-targeted mice lacking NF-E2, a transcription factor critical for megakaryopoieis, is not caused by a loss of platelet-dependent hemostatic function. In addition, we demonstrate (iv) that fibrinogen can confer physiologically relevant hemostatic function in the absence of platelets, but that a complete loss of hemostatic capacity results if a combined absence of these components is genetically imposed. These findings support the notion that the function of coagulation factors for in utero development and pregnancy is mechanistically distinct from their ability to mediate the formation of hemostatic platelet-fibrin(ogen) aggregates.

Arterial occlusive disorders are a leading cause of human morbidity. We hypothesized that ectopic expression of fibrinolytic proteins in platelets could be used to favorably alter the hemostatic balance at sites of thrombosis. To test our hypothesis, we directed murine urokinase-type plasminogen activator transgene expression to platelets using a platelet factor 4 promoter. Urokinase was selectively expressed and stored in the platelets of these mice. These transgenic mice had altered platelet biology and a bleeding diathesis similar to that seen in patients with Quebec platelet disorder, affirming the role of ectopic urokinase expression as the etiology of this inherited disease. These mice were resistant to the development of occlusive carotid artery thrombosis in the absence of systemic fibrinolysis and displayed rapid resolution of pulmonary emboli. Moreover, transfusion of urokinase-expressing platelets into wild-type mice prevented formation of occlusive arterial thrombi. These studies show the feasibility of delivering fibrinolytic agents to sites of incipient thrombus formation through selective storage in platelets and offer a new strategy to prevent thrombosis and hemorrhage.