Procoagulant platelet activation is a hallmark of venous thrombosis in human individuals and mice. (A) Scheme of clinical cohort and performed imaging entities to confirm DVT or PE. (B) Quantification of systemic D-dimer levels, leukocyte counts, percentage neutrophils of leukocytes, percentage platelet-neutrophil aggregates (PNA) of circulating neutrophils, circulating procoagulant platelets (PPs), and platelet surface expression of activated GPIIBIIIA and P-selectin in patients with suspected DVT or PE, comparing those without (black) vs those with confirmed thrombosis or thromboembolism (red). Student t-test, 2-tailed, unpaired. Gray boxes indicate normal range. (C) Representative specimen of interventionally retrieved human pulmonary embolus (white line) that was retrieved using a transfemoral mechanical thrombectomy device. Scale bar = 5 cm. See supplemental Figure 2A and supplemental Video 1 for further imaging data on the same patient. (D) Representative IF staining of a human pulmonary embolus retrieved through interventional thrombectomy and stained for CD42b, fibrinogen, and phosphatidylserine (PS). Scale bar = 10 μm (left) and 3 μm (right panel). See supplemental Figure 2C-D for individual channels, further examples, and unstained controls. (E) Experimental scheme of inferior vena cava (IVC) stenosis model. (F) Quantification of circulating PPs in wild-type mice undergoing the stenosis-induced vena cava thrombosis model, comparing mice without vs those with evidence of caval thrombosis. Student t-test, 2-tailed, unpaired. (G) Linear regression analysis to correlate thrombus weight in mice with percentage circulating PPs or the indicated platelet surface markers. (H) Representative IF staining of a murine IVC thrombus stained for CD42b, fibrinogen, and PS. The highlighted, framed area is shown in split channels on the right-hand side. Scale bars = 25 μm (left panel), 15 μm (other panels). See supplemental Figure 2G for unstained controls. (I) Scheme of flow reduction–induced mesenteric vein thrombosis and live-imaging approach and representative confocal overview (generated through maximum intensity projection) of a ligated mesenteric vein; the white silk thread is visualized in the upper part of the image. Scale bar = 50 μm. (J) Representative confocal image of a developing thrombus in a mesenteric vein 1 hour after flow reduction. Right: split channels showing a procoagulant platelet derived from the highlighted area (white dashed line) of the developing thrombus. Scale bars = left 50, right 5 μm. See supplemental Video 2 for correlating live microscopy. (K) Scheme of flow reduction–induced IVC thrombosis and live-imaging approach as well as representative confocal overview of a developing thrombus. Right: split channels showing a procoagulant platelet derived from the highlighted area (white dashed line) of the developing thrombus. Micrographs below the CD42b channel highlight the colocalization of CD42b and PS along with local binding of fibrin(ogen). Pseudocolored image depicts areas of colocalization between CD42b and PS. Scale bars = left 50, right 10 μm. White arrow indicates the direction of blood flow. Scale bar = 50 μm. (L) Representative confocal image of a developing thrombus in an IVC ≈3 hours after flow reduction. Scale bar = 50 μm. See supplemental Video 3 for correlating live microscopy. Graph to right: normalized intensity profiles of CD41, fibrin(ogen), and PS, corresponding to red line in the image to the left. (M) Explanatory scheme and quantification of triple-positive areas vs Fbg/PS-only positive areas in IVC thrombi, representing fibrin(ogen) recruitment of procoagulant platelets vs fibrin(ogen) recruitment by leukocytes and/or endothelial cells. Individual data points correspond to randomly acquired images of IVC thrombi (n = 30) derived from n = 4 mice. Unless otherwise stated, all statistical tests were Student t-test, 2-tailed, unpaired. P values corresponding to asterisks: ∗P < .05, ∗∗P < .01, ∗∗∗P < .005.

Procoagulant platelet activation is a hallmark of venous thrombosis in human individuals and mice. (A) Scheme of clinical cohort and performed imaging entities to confirm DVT or PE. (B) Quantification of systemic D-dimer levels, leukocyte counts, percentage neutrophils of leukocytes, percentage platelet-neutrophil aggregates (PNA) of circulating neutrophils, circulating procoagulant platelets (PPs), and platelet surface expression of activated GPIIBIIIA and P-selectin in patients with suspected DVT or PE, comparing those without (black) vs those with confirmed thrombosis or thromboembolism (red). Student t-test, 2-tailed, unpaired. Gray boxes indicate normal range. (C) Representative specimen of interventionally retrieved human pulmonary embolus (white line) that was retrieved using a transfemoral mechanical thrombectomy device. Scale bar = 5 cm. See supplemental Figure 2A and supplemental Video 1 for further imaging data on the same patient. (D) Representative IF staining of a human pulmonary embolus retrieved through interventional thrombectomy and stained for CD42b, fibrinogen, and phosphatidylserine (PS). Scale bar = 10 μm (left) and 3 μm (right panel). See supplemental Figure 2C-D for individual channels, further examples, and unstained controls. (E) Experimental scheme of inferior vena cava (IVC) stenosis model. (F) Quantification of circulating PPs in wild-type mice undergoing the stenosis-induced vena cava thrombosis model, comparing mice without vs those with evidence of caval thrombosis. Student t-test, 2-tailed, unpaired. (G) Linear regression analysis to correlate thrombus weight in mice with percentage circulating PPs or the indicated platelet surface markers. (H) Representative IF staining of a murine IVC thrombus stained for CD42b, fibrinogen, and PS. The highlighted, framed area is shown in split channels on the right-hand side. Scale bars = 25 μm (left panel), 15 μm (other panels). See supplemental Figure 2G for unstained controls. (I) Scheme of flow reduction–induced mesenteric vein thrombosis and live-imaging approach and representative confocal overview (generated through maximum intensity projection) of a ligated mesenteric vein; the white silk thread is visualized in the upper part of the image. Scale bar = 50 μm. (J) Representative confocal image of a developing thrombus in a mesenteric vein 1 hour after flow reduction. Right: split channels showing a procoagulant platelet derived from the highlighted area (white dashed line) of the developing thrombus. Scale bars = left 50, right 5 μm. See supplemental Video 2 for correlating live microscopy. (K) Scheme of flow reduction–induced IVC thrombosis and live-imaging approach as well as representative confocal overview of a developing thrombus. Right: split channels showing a procoagulant platelet derived from the highlighted area (white dashed line) of the developing thrombus. Micrographs below the CD42b channel highlight the colocalization of CD42b and PS along with local binding of fibrin(ogen). Pseudocolored image depicts areas of colocalization between CD42b and PS. Scale bars = left 50, right 10 μm. White arrow indicates the direction of blood flow. Scale bar = 50 μm. (L) Representative confocal image of a developing thrombus in an IVC ≈3 hours after flow reduction. Scale bar = 50 μm. See supplemental Video 3 for correlating live microscopy. Graph to right: normalized intensity profiles of CD41, fibrin(ogen), and PS, corresponding to red line in the image to the left. (M) Explanatory scheme and quantification of triple-positive areas vs Fbg/PS-only positive areas in IVC thrombi, representing fibrin(ogen) recruitment of procoagulant platelets vs fibrin(ogen) recruitment by leukocytes and/or endothelial cells. Individual data points correspond to randomly acquired images of IVC thrombi (n = 30) derived from n = 4 mice. Unless otherwise stated, all statistical tests were Student t-test, 2-tailed, unpaired. P values corresponding to asterisks: ∗P < .05, ∗∗P < .01, ∗∗∗P < .005.

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