Irina Pleines

Cell Adhesion Mechanisms in Platelets

At sites of vascular injury, platelets come into contact with the subendothelial extracellular matrix which triggers their activation and the formation of a hemostatic plug. This process is crucial for normal hemostasis, but may also lead to pathological thrombus formation causing diseases such as myocardial infarction or stroke. The initial capture of flowing platelets is mediated by the interaction of the glycoprotein (GP) Ib-V-IX complex with von Willebrand factor (vWF) immobilized on exposed collagens. This interaction allows the binding of the collagen receptor GPVI to its ligand and to initiate cellular activation, a process that is reinforced by locally produced thrombin and soluble mediators released from platelets. These events lead to the shift of &bgr;1 and &bgr;3 integrins on the platelet surface from a low to a high affinity state, thereby enabling them to bind their ligands and to mediate firm adhesion, spreading, coagulant activity, and aggregation. This review summarizes the most important structural and functional properties of these adhesion receptors and briefly discusses their potential as targets for antithrombotic therapy.

Orai1 (CRACM1) is the platelet SOC channel and essential for pathological thrombus formation

Platelet activation and aggregation at sites of vascular injury are essential for primary hemostasis, but are also major pathomechanisms underlying myocardial infarction and stroke. Changes in [Ca(2+)](i) are a central step in platelet activation. In nonexcitable cells, receptor-mediated depletion of intracellular Ca(2+) stores triggers Ca(2+) entry through store-operated calcium (SOC) channels. STIM1 has been identified as an endoplasmic reticulum (ER)-resident Ca(2+) sensor that regulates store-operated calcium entry (SOCE) in immune cells and platelets, but the identity of the platelet SOC channel has remained elusive. Orai1 (CRACM1) is the recently discovered SOC (CRAC) channel in T cells and mast cells but its role in mammalian physiology is unknown. Here we report that Orai1 is strongly expressed in human and mouse platelets. To test its role in blood clotting, we generated Orai1-deficient mice and found that their platelets display severely defective SOCE, agonist-induced Ca(2+) responses, and impaired activation and thrombus formation under flow in vitro. As a direct consequence, Orai1 deficiency in mice results in resistance to pulmonary thromboembolism, arterial thrombosis, and ischemic brain infarction, but only mild bleeding time prolongation. These results establish Orai1 as the long-sought platelet SOC channel and a crucial mediator of ischemic cardiovascular and cerebrovascular events.

The calcium sensor STIM1 is an essential mediator of arterial thrombosis and ischemic brain infarction

Platelet activation and aggregation are essential to limit posttraumatic blood loss at sites of vascular injury but also contributes to arterial thrombosis, leading to myocardial infarction and stroke. Agonist-induced elevation of [Ca2+]i is a central step in platelet activation, but the underlying mechanisms are not fully understood. A major pathway for Ca2+ entry in nonexcitable cells involves receptor-mediated release of intracellular Ca2+ stores, followed by activation of store-operated calcium (SOC) channels in the plasma membrane. Stromal interaction molecule 1 (STIM1) has been identified as the Ca2+ sensor in the endoplasmic reticulum (ER) that activates Ca2+ release–activated channels in T cells, but its role in mammalian physiology is unknown. Platelets express high levels of STIM1, but its exact function has been elusive, because these cells lack a normal ER and Ca2+ is stored in a tubular system referred to as the sarcoplasmatic reticulum. We report that mice lacking STIM1 display early postnatal lethality and growth retardation. STIM1-deficient platelets have a marked defect in agonist-induced Ca2+ responses, and impaired activation and thrombus formation under flow in vitro. Importantly, mice with STIM1-deficient platelets are significantly protected from arterial thrombosis and ischemic brain infarction but have only a mild bleeding time prolongation. These results establish STIM1 as an important mediator in the pathogenesis of ischemic cardio- and cerebrovascular events.

Loss of talin1 in platelets abrogates integrin activation, platelet aggregation, and thrombus formation in vitro and in vivo

Platelet adhesion and aggregation at sites of vascular injury are essential for normal hemostasis but may also lead to pathological thrombus formation, causing diseases such as myocardial infarction or stroke. Heterodimeric receptors of the integrin family play a central role in the adhesion and aggregation of platelets. In resting platelets, integrins exhibit a low affi nity state for their ligands, and they shift to a high affi nity state at sites of vascular injury. It has been proposed that direct binding of the cytoskeletal protein talin1 to the cytoplasmic domain of the integrin subunits is necessary and suffi cient to trigger the activation of integrins to this high affi nity state, but direct in vivo evidence in support of this hypothesis is still lacking. Here, we show that platelets from mice lacking talin1 are unable to activate integrins in response to all known major platelet agonists while other cellular functions are still preserved. As a consequence, mice with talindefi cient platelets display a severe hemostatic defect and are completely resistant to arterial thrombosis. Collectively, these experiments demonstrate that talin is required for inside-out activation of platelet integrins in hemostasis and thrombosis.

Platelet adhesion and activation mechanisms in arterial thrombosis and ischaemic stroke

Summary.  Platelet adhesion, activation and aggregation on the exposed subendothelial extracellular matrix (ECM) are essential for haemostasis, but may also lead to occlusion of diseased vessels. Binding of the glycoprotein (GP)Ib‐V‐IX complex to immobilised von Willebrand factor (VWF) initiates adhesion of flowing platelets to the ECM, and thereby enables the collagen receptor GPVI to interact with its ligand and to mediate platelet activation. This process is reinforced by locally produced thrombin and platelet‐derived secondary mediators, such as adenosine diphosphate (ADP) and thromboxane A2 (TxA2). Together, these events promote a shift of β1 and β3 integrins from a low to a high affinity state for their ligands through ‘inside‐out’ signalling allowing firm platelet adhesion and aggregation. Formed platelet aggregates are stabilised by fibrin formation and signalling events between adjacent platelets involving multiple platelet receptors, such as the newly discovered C‐type lectin‐like receptor 2 (CLEC‐2). While occlusive thrombus formation is the principal pathogenic event in myocardial infarction, the situation is more complex in ischaemic stroke where infarct development often progresses despite sustained early reperfusion of previously occluded major intracranial arteries, a process referred to as ‘reperfusion injury’. Increasing experimental evidence now suggests that early platelet adhesion and activation events, orchestrate a ‘thrombo‐inflammatory’ cascade in this setting, whereas platelet aggregation and thrombus formation are not required. This review summarises recent developments in understanding the principal platelet adhesion receptor systems with a focus on their involvement in arterial thrombosis and ischaemic stroke models.

Factor XIIa Inhibitor Recombinant Human Albumin Infestin-4 Abolishes Occlusive Arterial Thrombus Formation Without Affecting Bleeding

Background— Blood coagulation is a tightly regulated process of sequentially activated serine proteases culminating in fibrin formation, which is critical for limiting posttraumatic blood loss but also may contribute to acute thrombotic diseases, most notably myocardial infarction and stroke. Recent studies with factor XII–deficient mice revealed that the factor XII–induced intrinsic coagulation pathway is essential for pathological thrombus formation but dispensable for hemostasis. Consequently, these findings led to the hypothesis that factor XII could be a promising pharmacological target for safe antithrombotic therapy. Methods and Results— The complementary DNA of the previously described factor XIIa inhibitor Infestin-4, cloned from the midgut of Triatoma infestans, was fused to recombinant human albumin (rHA) and analyzed in vitro. The resulting protein rHA-Infestin-4 specifically inhibits factor XIIa and causes prolonged activated partial thromboplastin time in human, mouse, and rat plasma. To assess its inhibitory potency in vivo, mice and rats were injected with rHA-Infestin-4 and challenged in pathological thrombus formation models. In addition, bleeding assays were performed. rHA-Infestin-4 completely abolished occlusive arterial thrombus formation in mice and rats while leaving hemostasis fully intact. Furthermore, rHA-Infestin-4 was highly protective in a murine model of ischemic stroke. Conclusion— These results identify rHA-Infestin-4 as a promising agent to achieve powerful protection from ischemic cardiovascular and cerebrovascular events without affecting hemostasis.

Megakaryocyte-specific RhoA deficiency causes macrothrombocytopenia and defective platelet activation in hemostasis and thrombosis

Vascular injury initiates rapid platelet activation that is critical for hemostasis, but it also may cause thrombotic diseases, such as myocardial infarction or ischemic stroke. Reorganizations of the platelet cytoskeleton are crucial for platelet shape change and secretion and are thought to involve activation of the small GTPase RhoA. In this study, we analyzed the in vitro and in vivo consequences of megakaryocyte- and platelet-specific RhoA gene deletion in mice. We found a pronounced macrothrombocytopenia in RhoA-deficient mice, with platelet counts of approximately half that of wild-type controls. The mutant cells displayed an altered shape but only a moderately reduced life span. Shape change of RhoA-deficient platelets in response to G(13)-coupled agonists was abolished, and it was impaired in response to G(q) stimulation. Similarly, RhoA was required for efficient secretion of α and dense granules downstream of G(13) and G(q). Furthermore, RhoA was essential for integrin-mediated clot retraction but not for actomyosin rearrangements and spreading of activated platelets on fibrinogen. In vivo, RhoA deficiency resulted in markedly prolonged tail bleeding times but also significant protection in different models of arterial thrombosis and in a model of ischemic stroke. Together, these results establish RhoA as an important regulator of platelet function in thrombosis and hemostasis.

Multiple alterations of platelet functions dominated by increased secretion in mice lacking Cdc42 in platelets

Platelet activation at sites of vascular injury is crucial for hemostasis, but it may also cause myocardial infarction or stroke. Cytoskeletal reorganization is essential for platelet activation and secretion. The small GTPase Cdc42 has been implicated as an important mediator of filopodia formation and exocytosis in various cell types, but its exact function in platelets is not established. Here, we show that the megakaryocyte/platelet-specific loss of Cdc42 leads to mild thrombocytopenia and a small increase in platelet size in mice. Unexpectedly, Cdc42-deficient platelets were able to form normally shaped filopodia and spread fully on fibrinogen upon activation, whereas filopodia formation upon selective induction of GPIb signaling was reduced compared with wild-type platelets. Furthermore, Cdc42-deficient platelets showed enhanced secretion of alpha granules, a higher adenosine diphosphate (ADP)/adenosine triphosphate (ATP) content, increased aggregation at low agonist concentrations, and enhanced aggregate formation on collagen under flow. In vivo, lack of Cdc42 resulted in faster occlusion of ferric chloride-injured arterioles. The life span of Cdc42-deficient platelets was markedly reduced, suggesting increased clearing of the cells under physiologic conditions. These data point to novel multiple functions of Cdc42 in the regulation of platelet activation, granule organization, degranulation, and a specific role in GPIb signaling.

Rac1 is essential for phospholipase C-γ2 activation in platelets

Platelet activation at sites of vascular injury is triggered through different signaling pathways leading to activation of phospholipase (PL) Cβ or PLCγ2. Active PLCs trigger Ca2+ mobilization and entry, which is a prerequisite for adhesion, secretion, and thrombus formation. PLCβ isoenzymes are activated downstream of G protein-coupled receptors (GPCRs), whereas PLCγ2 is activated downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors, such as the major platelet collagen receptor glycoprotein (GP) VI or CLEC-2. The mechanisms underlying PLC regulation are not fully understood. An involvement of small GTPases of the Rho family (Rho, Rac, Cdc42) in PLC activation has been proposed but this has not been investigated in platelets. We here show that murine platelets lacking Rac1 display severely impaired GPVI- or CLEC-2-dependent activation and aggregation. This defect was associated with impaired production of inositol 1,4,5-trisphosphate (IP3) and intracellular calcium mobilization suggesting inappropriate activation of PLCγ2 despite normal tyrosine phosphorylation of the enzyme. Rac1−/− platelets displayed defective thrombus formation on collagen under flow conditions which could be fully restored by co-infusion of ADP and the TxA2 analog U46619, indicating that impaired GPVI-, but not G-protein signaling, was responsible for the observed defect. In line with this, Rac1−/− mice were protected in two collagen-dependent arterial thrombosis models. Together, these results demonstrate that Rac1 is essential for ITAM-dependent PLCγ2 activation in platelets and that this is critical for thrombus formation in vivo.

Deficiency of the Tetraspanin CD63 Associated with Kidney Pathology but Normal Lysosomal Function

ABSTRACT CD63 is a member of the tetraspanin superfamily that constitutes a main component of the lysosomal membrane. In mice, two CD63 gene loci are present, with only one of these two being functional. We generated and analyzed mice deficient for active CD63. Disruption of CD63 results in a complete loss of CD63 protein expression. Despite its abundance in late endosomes/lysosomes, the lack of CD63 does not cause obvious endosomal/lysosomal abnormalities. CD63 knockout mice are viable and fertile without gross morphological abnormalities in the majority of tissues. No alterations in the populations of immune cells and only minor differences in platelet function were observed. This suggests that the lack of CD63 could be successfully compensated for, most likely by other tetraspanins. However, CD63 deficiency leads to an altered water balance. CD63 knockout mice show an increased urinary flow, water intake, reduced urine osmolality, and a higher fecal water content. In principle cells of the collecting duct of CD63-deficient mice, abnormal intracellular lamellar inclusions were observed. This indicates that the sorting of apical transport proteins might be impaired in these cells. CD63 knockout mice provide an important tool for analyzing the various postulated functions of CD63 in vivo.