Direct red blood cell effect on thrombosis is dependent on the interaction of tissue factor and calcium with membrane phosphatidylserine

Abstract

Background: Prior literature has implicated red blood cells (RBCs) in the initiation of thrombosis and suggests that posttransfusion hypercoagulability may occur secondary to the effects of RBCs. Elevated serum tissue factor is a known sequelae of acute trauma. Phosphatidylserine (PS) is a prothrombotic phospholipid present within the RBC cell membrane. We hypothesized that RBC aggregation is dependent on the interaction between RBC membrane bound (exposed) PS, extracellular calcium, and tissue factor.

Methods: Human whole blood (WB) was separated into components, including RBCs and platelet-rich plasma (PRP). Whole blood, PRP, and RBCs underwent impedance aggregometry utilizing arachidonic acid (AA), ADP, collagen, calcium, and tissue factor (TF)-based agonists. Red blood cells then underwent impedance aggregometry utilizing combined calcium and TF agonists. Red blood cells were pretreated with Annexin V, a known PS blocking agent, and underwent impedance aggregometry with combined calcium and TF agonists to determine if the mechanism of calcium/TF-induced RBC aggregability is dependent on PS. Red blood cells treated with calcium, TF, calcium+TF, and pre-treated with Annexin V followed by calcium+TF were perfused through an in vitro model of pulmonary microcirculatory flow.

Results: Red blood cell aggregation was significantly higher than that of WB and PRP when utilizing a TF agonist, an effect unique to TF. The combination of calcium and TF demonstrated significantly higher RBC aggregation than either agonist alone. Pretreatment with Annexin V resulted in a significantly reduced aggregability of RBC following treatment with TF + calcium. Red blood cells aged to 42 days did not exhibit significant change in aggregation. Exposure to calcium and TF significantly reduced time to thrombosis of RBCs perfused through a pulmonary microcirculatory model.

Conclusion: Treatment with both TF and calcium synergistically induces RBC aggregation. Phosphatidylserine appears to play an integral role in the TF/calcium-based, age-independent RBC aggregation response. Red blood cells treated with TF + calcium exhibit more rapid thrombus formation in an in vitro model of pulmonary microcirculatory perfusion.

Figure 1.

Following serial centrifugation, platelet count was obtained on WB, PRP, and RBC samples from donated WB samples. Isolated PRP had significantly elevated platelet count compared to both WB (p<0.01) and RBC (p<0.01). RBC also demonstrated significantly lower platelet count than WB (p<0.01), demonstrating adequate removal of platelets from isolated RBC.

Figure 2.

WB, PRP, and RBC (n=4 per group) were isolated and tested with impedance aggregometry utilizing agonists ADP, ASPI, collagen, CaCl₂, and rTF. ADP demonstrated higher aggregation in WB than in RBC (p=0.02). There was no significant difference in aggregation utilizing the ASPI agonist. Collagen demonstrated increased aggregation in both WB and RBC compared to PRP (p<0.01 and p=0.04, respectively). CaCl₂ demonstrated increased aggregation in WB when compared to PRP (p=0.03). rTF demonstrated increased aggregation in WB compared to PRP (p<0.01), as well as RBC compared to PRP and WB (p<0.01 for both groups).

Figure 3.

RBCs isolated from donated units of WB (n=4) and analyzed with impedance aggregometry demonstrated higher aggregation when both CaCl₂ and rTF were used, compared to either CaCl₂ (p<0.01) or rTF (p=0.02) alone. (A) RBCs isolated from trauma patient WB samples (n=10) and analyzed with impedance aggregometry demonstrated higher aggregation when both CaCl₂ and rTF were used, compared to either CaCl₂ (p<0.01) or rTF (p<0.01) alone. (B)

Figure 4.

RBCs isolated from donated units of WB (n=4) demonstrated persistent aggregation when exposed to both CaCl₂ and rTF following storage for 14 days, 21 days, and 42 days when compared to fresh (day 2) stored pRBC.

Figure 5.

RBCs isolated from donated units of WB (n=4) demonstrated decreased aggregation when treated with Annexin V (ANX5) compared to untreated RBCs utilizing agonists CaCl₂ and rTF agonists at age day 21 (p<0.01).

Figure 6.

RBCs isolated from WB exposed to agonist CaCl₂+rTF demonstrated a significantly lower time to thrombosis than treatment with CaCl₂ alone (p<0.01) or rTF alone (p=0.01). RBCs incubated with Annexin V prior to treatment with agonist CaCl₂+rTF demonstrated a longer time to thrombosis than RBCs without Annexin V exposed to CaCl₂+rTF (p=0.02). RBCs treated with CaCl₂ alone demonstrated a longer time to thrombosis than RBCs incubated with Annexin V prior to exposure to agonists CaCl₂+rTF (p=0.02).