Figure 1.
Deletion of the GPVI and FcRγ receptors, but not the α2β1 integrin alters collagen-induced intravascular thrombosis and pulmonary embolism.
(A) The percentage decrease in platelet count 3 minutes after injection of Type I collagen (25ug) and epinephrine (1ug) was determined for wild type (WT), α2β1−/−, GPVI−/−, FcRγ−/−, α2β1−/−/GPVI−/−, and α2β1−/−/FcRγ−/− mice on the mixed genetic background (129/SvJ × C57BL/6). (B) The number of pulmonary thrombi after injection of Type I collagen was determined for wild type (WT), α2β1−/−, GPVI−/−, FcRγ−/−, α2β1−/−/GPVI−/−, and α2β1−/−/FcRγ−/− mice on a 129/SvJ × C57BL/6 background. Thrombi were recorded per mm2 in 6 random 20X fields.
Figure 2.
Deletion of either the α2β1 integrin or the GPVI subunit, but not the FcRγ receptor delays carotid artery thrombosis.
The length of time to complete arterial occlusion following photochemical injury of the carotid artery was recorded in wild type (WT), α2β1−/−, FcRγ−/−, and GPVI−/− mice on a mixed genetic background (129/SvJ × C57BL/6). The values represent the mean ± SD for WT (n = 12), α2β1−/− (n = 15), GPVI−/− (n = 7), or FcRγ−/− (n = 7) animals.
Figure 3.
The in vivo thrombotic differences are independent of genetic background.
(A) The platelet count decrement (% change from baseline) following intravenous injection of Type I collagen (25ug) and epinephrine (1ug) into wild type (WT) (n = 7), GPVI−/− (n = 7), FcRγ−/− (n = 9) or α2β1−/− (n = 11) mice on a pure C57BL/6 background was determined. (B) The number of thrombi observed in the lungs at 3 minutes after injection of Type I collagen (25ug) and epinephrine (1ug) was determined for wild type (WT), α2β1−/−, GPVI−/−, FcRγ−/− mice on the C57BL/6 background. Thrombi were recorded per mm2 in 6 random 20X fields.
Figure 4.
Differences in thrombotic occlusion of the carotid artery are independent of genetic background.
The impact of genetic background on photochemically induced endothelial injury on carotid artery occlusion was also evaluated in α2β1−/−, FcRγ−/−, GPVI−/− and wild type mice on a pure C57BL/6 genetic background. The length of time to complete vessel occlusion following photochemical injury was measured in wild type, α2β1−/−, FcRγ−/−, and GPVI−/− mice on the pure genetic background (C57BL/6). The values represent the mean ± SD of WT (n = 10), α2β1−/− (n = 10), GPVI−/− (n = 5), or FcRγ−/− (n = 8).
Figure 5.
Type I collagen-stimulated platelet adhesion, aggregation, and spreading.
(A and B) Purified platelets isolated from wild type (WT), α2β1−/−, GPVI−/−, or FcRγ−/− mice were assayed for adhesion to Type I collagen (CNI), or BSA for 60 minutes (A) or over a time course of 60 minutes (B) in vitro. Results represent percentages of adherent platelets (mean of 3 independent experiments performed in triplicate). (C) Purified platelets isolated from wild type (WT), α2β1−/−, GPVI−/−, or FcRγ−/− mice were assayed for adhesion to Type I collagen (CNI) in the presence or absence of inhibitory anti-α2β1−/− antibody or BSA for 60 minutes in vitro. Results represent percentages of adherent platelets (mean of 2 independent experiments performed in triplicate). (D) Scanning electron micrographs detail wild type (WT), GPVI−/−, or FcRγ−/− mouse platelets when adherent to collagen I (CNI) for 60 minutes. Platelets from α2β1−/− mice failed to adhere and therefore were not observed.
Figure 6.
Protein phospho-tyrosine analyses of wild type (WT), GPVI−/−, and FcRγ−/− mouse platelets.
(A) Purified platelets from WT, GPVI−/−, or FcRγ−/− mice were either untreated or stimulated with 10 µg/mL collagen I In the presence of 2 mM MgCl2 for 1 minute followed by immunoblot analysis using antibodies against phospho-tyrosine (pTyr) or actin. Protein bands of interest are indicated with arrows at molecular weights of about 25 and 72 kDa. Quantification of the 25 and 72 kDa pTyr bands were performed by densitometry and normalized to actin. (B) WT, GPVI−/−, or FcRγ−/− mouse platelets stimulated with CNI were analyzed for phosphorylated Syk (pSyk) and total Syk by immunoblot. In the presence of 2 mM MgCl2, control platelets or platelets treated with 10 µg/mL CNI for 1 minute, were lysed, and analyzed by Western blot using antibodies for phospho-Syk (Tyr525/526), total Syk protein, or total phospho-tyrosine. Quantification of the pSyk and total Syk was performed by densitometry and the value of pSyk normalized to total Syk. (C) Western blot analysis of WT, GPVI−/−, or FcRγ−/− mouse platelets for phosphorylated RhoGDI and total RhoGDI following CNI stimulation as described. Quantification of the pRhoGDI and total RhoGDI was performed and the value of pRhoGDI normalized to total RhoGDI. (D) Western blot analysis of unstimulated WT, GPVI−/−, or FcRγ−/− mouse platelets for total FcRγ protein or actin expression.