Impaired “outside-in” integrin αIIbβ3 signaling and thrombus stability in TSSC6-deficient mice

Abstract

We investigated the role of the hematopoietic-specific tetraspanin superfamily member, TSSC6, in platelet function using wild-type mice and TSSC6-deficient mice. TSSC6 is expressed on the surface of murine platelets and is up-regulated by thrombin stimulation, indicating an intracellular pool of TSSC6. Immunoprecipitation/Western blot studies reveal a constitutive physical association of TSSC6 with the integrin ␣ IIb ␤ 3 complex under strong detergent conditions. In vivo evaluation of hemostasis by tail bleeding revealed increased bleeding time, volume of blood lost, and evidence of tail rebleeds in TSSC6 null mice, indicating unstable hemostasis. Using ex vivo techniques, we showed that TSSC6-deficient platelets exhibited impaired kinetics of clot retraction, platelet aggregation at lower doses of PAR-4, and collagen and platelet spreading on fibrinogen in the presence of normal integrin ␣ IIb ␤ 3 expression. TSSC6-deficient platelets showed normal alpha granule secretion, normal "insideout" integrin ␣ IIb ␤ 3 signaling (fluorescein isothiocyanate [FITC]-fibrinogen and JON/A binding), and normal platelet adhesion on fibrinogen. Furthermore, we show that absence of platelet TSSC6 affects the secondary stability of arterial thrombi in vivo upon vascular injury. These data demonstrate that TSSC6 appears to regulate integrin ␣ IIb ␤ 3 "outside-in" signaling events in platelets and is necessary for stability of arterial thrombi in vivo. (Blood.

CEACAM1 negatively regulates platelet-collagen interactions and thrombus growth in vitro and in vivo

“…30 The only exception was that platelets were labeled with either preimmune rabbit serum (1:500), rabbit anti-mouse CEACAM1 2457 serum (1:500) or FITC-conjugated antibody to P-selectin (10 g/mL), and where appropriate, then labeled with swine anti-rabbit F(abЈ) 2 FITC-conjugated antibody (1:200; Dako).…”

“…A strip of filter paper (4 mm) was dipped in a 462 mM (7.5% wt/vol) FeCl 3 solution for 3 seconds, applied to a 2-to 5-mm length of arteriole for 4 minutes and then removed. 30,31 Z-stack images of the vessel were taken over 5 2-minute cycles (determined as the time the vessel begins to occlude). The rendered z-stack images were deconvolved with AxioVision Rel 4.6 software (Carl Zeiss), to produce a 3-dimensional image, and the volume of the thrombi were determined from the area and height of the clot.…”

“…The ferric chloride (FeCl 3 ) oxidative injury model was used to induce formation of free radicals to disrupt the vascular endothelium in mesenteric arterioles. 30 Platelet thrombus formation was monitored in real time by assessing the accumulation of fluorescently labeled platelets. After application of FeCl 3 to the mesentery, platelets in both wild-type and ceacam1 Ϫ/Ϫ mice rapidly commenced interacting with the injured vessel wall and within 10 to 15 minutes after the injury, vessel occlusion occurred.…”

Tetraspanin Tspan9 regulates platelet collagen receptor GPVI lateral diffusion and activation

“…To date five tetraspanins have been investigated on platelets using knockout mice: CD9, CD63, CD82, CD151 and Tspan32. Mice deficient in either CD151 or Tspan32 have similar phenotypes, namely a partial defect in platelet aggregation and haemostasis that is proposed to be due to impaired outside-in signalling of the major platelet integrin αIIbβ3 [19–21]. Conversely, deficiency in either CD9 or CD63 causes a mildly hyperactive platelet phenotype without affecting haemostasis [22, 23].…”

The Tetraspanin CD9 Localizes to Platelet-Platelet Contacts and Regulates Thrombus Stability

“…Inactivation of the tetraspanin TALLA-1 causes X-linked mental retardation (Zemni 2000); CD9 and CD81 knock-out mice have reduced fertility, with double knock-out mice being completely infertile (Kaji 2000, LaNaour 2000, Miyado 2000); and CD37 knock-out mice have reduced immune response. In the platelet system, CD151 or TSSC-6 knock-out mice have impaired integrin outside-in signaling (Goschnick 2006, Lau 2004.…”