T. Kent Gartner

Platelet glycoprotein Ibα supports experimental lung metastasis

The platelet paradigm in hemostasis and thrombosis involves an initiation step that depends on platelet membrane receptors binding to ligands on a damaged or inflamed vascular surface. Once bound to the surface, platelets provide a unique microenvironment supporting the accumulation of more platelets and the elaboration of a fibrin-rich network produced by coagulation factors. The platelet-specific receptor glycoprotein (GP) Ib-IX, is critical in this process and initiates the formation of a platelet-rich thrombus by tethering the platelet to a thrombogenic surface. A role for platelets beyond the hemostasis/thrombosis paradigm is emerging with significant platelet contributions in both tumorigenesis and inflammation. We have established congenic (N10) mouse colonies (C57BL/6J) with dysfunctional GP Ib-IX receptors in our laboratory that allow us an opportunity to examine the relevance of platelet GP Ib-IX in syngeneic mouse models of experimental metastasis. Our results demonstrate platelet GP Ib-IX contributes to experimental metastasis because a functional absence of GP Ib-IX correlates with a 15-fold reduction in the number of lung metastatic foci using B16F10.1 melanoma cells. The results demonstrate that the extracellular domain of the α-subunit of GP Ib is the structurally relevant component of the GP Ib-IX complex contributing to metastasis. Our results support the hypothesis that platelet GP Ib-IX functions that support normal hemostasis or pathologic thrombosis also contribute to tumor malignancy.

Platelet plasma membrane lectin activity

Abstract The lectin activity of human platelet and erythrocyte membranes was evaluated using trypsinized, formalinized erythrocytes from eight species. Platelet membranes had the greatest lectin activity against cow erythrocytes, but also had significant activity against human, sheep, electric eel, and rabbit erythrocytes. In contrast, erythrocyte membranes only had low lectin activity against electric eel erythrocytes with no activity against the other types of erythrocytes tested. The platelet membrane lectin activity was found to reside in protein molecules on the external surface of the platelet plasma membrane. The lectin activity of platelet membranes was inhibited by amino sugars and some basic amino acids: N-acetylated amino sugars and other neutral sugars were without effect. These results demonstrate that the external surface of the platelet plasma membrane has a specific lectin activity.

Thrombolectin: A lectin isolated from Bothrops atrox venom

Venom from the reptile Bothrops atrox can convert plasma fibrinogen into fibrin and induce platelet aggregation [l-3 1. Each activity is attributed to a distinct serine protease isolated from the venom: batroxobin [l] ( or reptilase) and thrombocytin [2,3]. Purified batroxobin can generate fibrin by hydrolysis of fibrinogen, yet is incapable of causing platelet a@regation El]. Conversely, thrombocytin induces platelet aggregation, but has very limited, if any, ability to convert ~b~nogen into fibrin [2,3f. Here we report that Bothrops atrox venom also contains a lectin which we have designated thrombolectin. This lectin is a disulfide-linked, dimeric protein of apparent monomer mol. wt -15 000. The activity of the lectin is inhibited maximally by P-galactosides and, in contrast to other galactolectins [4], is calciumdependent and inhibited by reducing agents.

Characterization of adhesion of "resting" and stimulated platelets to fibrinogen and its fragments.

Abstract Adhesion of resting and stimulated platelets to immobilized fibrinogen (Fg) was characterized using various forms of Fg, receptor peptide mimics, and antibodies to glycoprotein (GP) IIb/IIIa and Fg. Resting platelets adhered to Fg, but to less than half the extent of the same platelets stimulated with epinephrine/ADP. The adhesion of resting and stimulated platelets to Fg was inhibited by a receptor peptide mimic (G13, a peptide corresponding to residues 300–312 of GPIIb), anti-GPIIb/IIIa antibodies, and a monoclonal antibody (4A5) against the carboxyl terminus of the γ chain of Fg. The results presented here demonstrate that the γ chain RGD platelet recognition sites are not required to mediate the adhesion of either stimulated or resting platelets to immobilized Fg. Although stimulated platelets can adhere extensively to monomeric Fg containing one functional γ chain, resting platelets require bivalent Fg containing two functional γ chains to mediate irreversible adhesion to Fg.

Roles of Purinergic Receptor P2Y, G Protein–Coupled 12 in the Development of Atherosclerosis in Apolipoprotein E–Deficient Mice

Objective—The aim of the study was to evaluate the role of purinergic receptor P2Y, G protein–coupled 12 (P2Y12), an ADP receptor, in the development of atherosclerotic lesions. Methods and Results—Apolipoprotein E–null mice were crossed with P2y12−/− mice to generate double knockout mice. The double knockout mice and the control apolipoprotein E–null mice were fed a high-fat diet for 20 weeks. Assessment of the atherosclerotic lesions in the control and double knockout mice demonstrated that P2Y12 deficiency caused a diminished lesion area, an increased fibrous content at the plaque site, and decreased monocyte/macrophage infiltration of the lesions. Polymerase chain reaction studies revealed that white blood cells do not express significant levels of P2Y12. Bone marrow transplantation experiments confirmed that P2Y12 expressed on platelets is a key factor responsible for atherosclerosis, but do not exclude a role of smooth muscle cell P2Y12. Supernatant fluid from activated P2y12+/+ but not P2y12−/− platelets was capable of causing monocyte migration. In vitro studies showed that platelet P2Y12 deficiency suppressed platelet factor 4 secretion and P-selectin expression. Further work demonstrated that platelet P2Y12, through inhibition of the cAMP/protein kinase A pathway, critically regulates the release of platelet factor 4, and thereby affects monocyte recruitment and infiltration. Conclusion—These results demonstrate that P2Y12 modulates atherogenesis, at least in part by augmenting inflammatory cell recruitment via regulation of platelet &agr;-granule release.

PTEN regulates collagen-induced platelet activation

Phosphatidylinositol 3-kinase (PI3K) has been shown to play an important role in collagen-induced platelet activation, but the role(s) of PTEN, a major regulator of the PI3K/Akt signaling pathway, has not been examined in platelets. Here, we report that Pten(-/-) mouse blood contains 25% more platelets than Pten(+/+) blood and that PTEN deficiency significantly shortened the bleeding time, increased the sensitivity of platelets to collagen-induced activation and aggregation, and enhanced phosphorylation of Akt at Ser473 in response to collagen. Furthermore, we found that PP2, and the combination of apyrase, indomethacin + 1B5, respectively, inhibited collagen-induced aggregation in both PTEN(+/+) and PTEN(-/-) platelets. In contrast, LY294002 (a PI3K inhibitor) prevented the aggregation of PTEN(+/+), but not PTEN(-/-), platelets. Therefore, PTEN apparently regulates collagen-induced platelet activation through PI3K/Akt-dependent and -independent signaling pathways.

Antibodies against a 23Kd heparin binding fragment of thrombospondin inhibit platelet aggregation

Antiserum against a 23Kd heparin binding fragment of thrombospondin inhibits the aggregation of platelets in response to ADP, collagen or thrombin. The antiserum inhibits the secretion-dependent second phase, but not the primary phase of aggregation of platelets responding to ADP. Although immune serum added during the second phase of ADP-induced aggregation causes some inhibition of secretion, it also causes reversal of aggregation to the level produced during primary aggregation. Since thrombospondin is the endogenous lectin of human platelets, these results support the conclusion that the endogenous lectin mediates, at least in part, the secretion-dependent aggregation of platelets. Our data suggest that the region of thrombospondin which contains the heparin binding domain(s) present in the 23Kd fragment play(s) a critical role in secretion-dependent aggregation of platelets.

Distinct Roles for Rap1b Protein in Platelet Secretion and Integrin αIIbβ3 Outside-in Signaling*

Background: Rap1b is a small G protein that is a key regulator for platelet activation. Results: Agonist-induced Rap1b activation plays a role in platelet secretion, and integrin outside-in signaling-mediated Rap1b activation is important in platelet spreading on fibrinogen and clot retraction. Conclusion: There are dual activation mechanisms of Rap1 that play distinct roles in platelet function. Significance: Learning two novel functions of Rap1b in platelets. 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,N′,N′-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 Rap1b-deficient 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 Rap1b as well as novel functions of Rap1b in platelet secretion and in integrin αIIbβ3 outside-in signaling.

Platelet P2Y12 Is Involved in Murine Pulmonary Metastasis

The involvement of platelets in tumor progression is well recognized. The depletion of circulating platelets or pharmacologic inhibitors of platelet activation decreases the metastatic potential of circulating tumor cells in metastasis mouse models. The platelet ADP receptor P2Y12 amplifies the initial hemostatic responses activated by a variety of platelet agonists and stabilizes platelet aggregation, playing a crucial role in granule secretion, integrin activation and thrombus formation. However, the relationship between P2Y12 and tumor progression is not clear. In our study, the Lewis Lung Carcinoma (LLC) spontaneous metastatic mouse model was used to evaluate the role of P2Y12 in metastasis. The results demonstrated that P2Y12 deficiency significantly reduced pulmonary metastasis. Further studies indicated that P2Y12 deficiency diminished the ability of LLC cells to induce platelet shape change and release of active TGFβ1 by a non-contact dependent mechanism resulting in a diminished, platelet-induced EMT-like transformation of the LLC cells, and that transformation probably is a prerequisite of LLC cell metastasis. Immunohistochemical analyses indicated an obvious P2Y12 deficiency related attenuation of recruitment of VEGFR1+ bone marrow derived cell clusters, and extracellular matrix fibronectin deposition in lungs, which presumably are required for pre-metastatic niche formation. In contrast to the LLC cells, non-epithelial melanoma B16 cells induced platelet aggregation in a cell number and P2Y12-dependent manner. Also, a platelet induced EMT-like transformation of B16 cells is dependent on P2Y12. In agreement with the LLC cell model, platelet P2Y12 deficiency also results in significantly less lung metastasis in the B16 melanoma experimental metastasis model. These results demonstrate that P2Y12 is a safe drug target for anti-thrombotic therapy, and that P2Y12 may serve as a new target for inhibition of tumor metastasis.

ADP-Stimulated Activation of Akt During Integrin Outside-In Signaling Promotes Platelet Spreading by Inhibiting Glycogen Synthase Kinase-3β

Objective—Integrins mediate platelet adhesion and transmit outside-in signals leading to platelet spreading. Phosphoinositide 3-kinases (PI3Ks) play a critical role in outside-in signaling and platelet spreading; however, the mechanisms of PI3K activation and function in outside-in signaling are unclear. We sought to determine the role of the Akt family of serine/threonine kinases and activation mechanisms of the PI3K/Akt pathway in outside-in signaling. Methods and Results—Akt inhibitors and Akt3 knockout inhibited platelet spreading on fibrinogen, indicating that Akt is important in integrin outside-in signaling. Akt inhibitors and Akt3 knockout also diminished integrin-dependent phosphorylation of glycogen synthase kinase-3&bgr;. Inhibition of glycogen synthase kinase-3&bgr; reversed the inhibitory effects of Akt3 knockout and inhibitors of Akt or PI3K on platelet spreading, indicating that glycogen synthase kinase-3&bgr; is a downstream target of Akt in outside-in signaling. Integrin-dependent activation of the PI3K-Akt pathway requires Src family kinase. Akt phosphorylation is also significantly inhibited in ADP receptor P2Y12 knockout platelets and further inhibited in P2Y12 knockout platelets treated with a P2Y1 antagonist. Consistently, P2Y12 knockout and P2Y1 inhibition together reduced platelet spreading. Conclusion—These results demonstrate that integrin outside-in signaling and platelet spreading requires Src family kinase–dependent and ADP receptor–amplified activation of the PI3K-Akt-GSK-3&bgr; pathway.