Activation of Piezo1 channels in compressed red blood cells augments platelet-driven contraction of blood clots

Abstract

Background: Piezo1 is a mechanosensitive cationic channel that boosts intracellular [Ca²⁺]_i. Compression of red blood cells (RBCs) during platelet-driven contraction of blood clots may cause the activation of Piezo1.

Objectives: To establish relationships between Piezo1 activity and blood clot contraction.

Methods: Effects of a Piezo1 agonist, Yoda1, and antagonist, GsMTx-4, on clot contraction in vitro were studied in human blood containing physiological [Ca²⁺]. Clot contraction was induced by exogenous thrombin. Activation of Piezo1 was assessed by Ca²⁺ influx in RBCs and with other functional and morphologic features.

Results: Piezo1 channels in compressed RBCs are activated naturally during blood clot contraction and induce an upsurge in the intracellular [Ca²⁺]_i, followed by phosphatidylserine exposure. Adding the Piezo1 agonist Yoda1 to whole blood increased the extent of clot contraction due to Ca²⁺-dependent volumetric shrinkage of RBCs and increased platelet contractility due to their hyperactivation by the enhanced generation of endogenous thrombin on activated RBCs. Addition of rivaroxaban, the inhibitor of thrombin formation, or elimination of Ca²⁺ from the extracellular space abrogated the stimulating effect of Yoda1 on clot contraction. The Piezo1 antagonist, GsMTx-4, caused a decrease in the extent of clot contraction relative to the control both in whole blood and in platelet-rich plasma. Activated Piezo1 in compressed and deformed RBCs amplified the platelet contractility as a positive feedback mechanism during clot contraction.

Conclusion: The results obtained demonstrate that the Piezo1 channel expressed on RBCs comprises a mechanochemical modulator of blood clotting that may be considered a potential therapeutic target to correct hemostatic disorders.

Keywords: Piezo1; blood clotting; clot contraction; platelets; red blood cells.

FIGURE 1

Effects of an activator (Yoda1) and inhibitor (GsMTx-4) of Piezo1 channels and a Ca²⁺ ionophore (A23187) on the contraction of whole blood clots. (A) The extent of clot contraction in the absence (control) and presence of Yoda1 (5 μM), GsMTx-4 (1 μM), or A23187 (15 μM) was measured in blood samples from independent donors stabilized with soybean trypsin inhibitor to maintain physiological [Ca²⁺]. Results are presented as the median and IQR (25th and 75th percentiles). The Mann–Whitney U-test was used for comparisons with the control. (B) Averaged kinetic curves of blood clot contraction measured in the absence (control, dark gray) and presence of Yoda1 (red), GsMTx-4 (green), or A23187 (blue). The curves’ colors match the colors of the corresponding bars shown in Figure 1A.

FIGURE 2

Effects of an activator (Yoda1) and inhibitor (GsMTx-4) of Piezo1 channels on contraction of clots formed in platelet-rich plasma. The extent of clot contraction in the absence (control) and presence of Yoda1 (5 μM) or GsMTx-4 (1 μM) was measured in plasma samples obtained from the blood of 5 independent donors. The blood was stabilized with soybean trypsin inhibitor to maintain physiological [Ca²⁺]. Results are presented as the median and IQR (25th and 75th percentiles). The Mann–Whitney U-test was used for comparisons with the control. n.s., not statistically significant.

FIGURE 3

Effect of the Piezo1 activator Yoda1 on the size of red blood cells (RBCs) within contracted clots made from Ca²⁺-containing whole blood samples. (A, B) Representative scanning electron microscopy images of the interior of contracted clots formed in the (A) absence and (B) presence of Yoda1 (5 μM), showing polyhedral RBCs (polyhedrocytes) that result from compressive deformation during platelet-driven clot contraction. Magnification bars: 5 μm. (C) The area occupied by individual RBCs (RBC size) was determined from scanning electron microscopy images corresponding to contracted clots in the absence (control, n = 209) and presence of Yoda1 (Yoda1, n = 215). The results are presented as the median and IQR (25th and 75th percentiles). The Mann–Whitney U-test was used for comparisons with the control. For the numerical data, see Supplementary Table S3.

FIGURE 4

Thrombin generation assay shows increased thrombin formation induced by Yoda1-activated red blood cells (RBCs). (A) Representative thrombin generation curves were obtained in recalcified kaolin-activated platelet-free plasma mixed with RBCs (5 × 10⁶ in 100 μL) pretreated with a vehicle (dimethyl sulfoxide [DMSO], gray curve) or with 5 μM Yoda1 (black curve). (B–E) Average parameters of the thrombin generation assay, namely the (B) peak thrombin concentration, (C) time to peak, (D) endogenous thrombin potential (ETP, area under the curve), and (E) lag time, in kaolin-activated platelet-free plasma mixed with RBCs pretreated with DMSO vs Yoda1 (5 μM). Results are presented as the average and SD (n = 5). The Student’s paired t-test was used for comparisons. For the numerical data, see Supplementary Table S5.

FIGURE 5

Enhanced procoagulant activity of Yoda1-activated red blood cells (RBCs). (A) Representative thromboelastography curves obtained in Ca²⁺-containing platelet-free plasma mixed with RBCs pretreated with a vehicle (dimethyl sulfoxide [DMSO], black curve) or with 5 μM Yoda1 (gray curve), illustrating shortening of the reaction time (R1 vs R2) and an increase in the rate of clot strengthening (α1 vs α2) in the presence of Yoda1-treated RBCs; the maximal clot firmness (MA1 and MA2) was almost unaffected. (B,C) Average thromboelastography parameters, namely the (B) reaction time, (C) clot strengthening, and (D) maximal clot firmness, in plasma mixed with RBCs pretreated with DMSO vs 5-μM Yoda1. Results are presented as the average and SD (n = 3). The Student’s paired t-test was used for comparisons. For the numerical data, see Supplementary Table S6. n.s., not statistically significant.

FIGURE 6

Effects of Yoda1 (5 μM) on blood clot contraction in the absence and presence of rivaroxaban (1 μM), an inhibitor of endogenous thrombin generation. Comparative experiments were performed in parallel on identical samples of whole blood obtained from 6 independent donors and stabilized with soybean trypsin inhibitor. Results are presented as the median and IQR (25th and 75th percentiles). The Mann–Whitney U-test was used for comparisons. n.s., not statistically significant.

FIGURE 7

Piezo1-mediated Ca²⁺ influx into compressed red blood cells during clot contraction. The plot shows comparative fluorescence intensity normalized by the concentration of hemoglobin (Hb) in the lysates of reconstituted blood clots containing red blood cells preloaded with Fluo-4, a Ca²⁺-sensitive dye (see Supplementary Materials and Methods for details). The clots from the same blood samples were formed in the following conditions: 1) a freely contracting clot; 2) an uncontracted clot in which platelet contractility was suppressed with 5 μM latrunculin A, an inhibitor of actin polymerization; 3) a contracting clot formed in the presence of 2 μM GsMTx-4, a Piezo1 inhibitor; 4) a contracting clot formed in the presence of 5 μM Yoda1, a Piezo1 activator; and 5) a contracting clot formed in the presence of 20 μM calcium ionophore A23187. The various types of clots were formed from the same blood samples and analyzed in parallel. Results are presented as the average and SD (n = 8). The Student’s paired t-test was used for comparisons. For the numerical data, see Supplementary Table S7. n.s., not statistically significant.

FIGURE 8

Proposed mechanism of self-amplification of blood clot contraction through activation of mechanosensitive Piezo1 ion channels in compressed and deformed red blood cells (RBCs). Platelet activation and blood clot formation were induced by the addition of exogenous thrombin. Activated platelets generate contractile forces that are transmitted to and distributed by the fibrin fibers. Platelet contraction and compaction of the fibrin network are accompanied by compressive deformation of RBCs to polyhedral shape (polyhedrocytes or piezocytes) and reduction in size. Mechanical deformation of RBCs causes activation of Piezo1 channels and Ca²⁺ influx. An increase in intracellular [Ca²⁺]_i activates scramblase, which mixes membrane phospholipids, causing externalization of phosphatidylserine. Phosphatidylserine forms a membrane matrix for the assembly of intrinsic tenase and prothrombinase, which catalyzes the conversion of prothrombin to thrombin under the action of factor Xa. The resulting endogenous thrombin diffuses inside the clot and additionally activates platelets by binding to PAR1 and PAR4 receptors. Enhancing platelet contractility further augments the compression of the blood clot.