Comprehensive Analysis of Procoagulant Platelets Exhibiting Features of Necrosis, Apoptosis and Platelet Activation

Summary

The present protocol provides a comprehensive set of procedures necessary to analyze procoagulant platelets, which exhibit overlapping features of necrosis, apoptosis, and platelet activation.

Abstract

Platelets circulating in the bloodstream are relatively quiescent but become "activated" upon encountering stimulants or "agonists" at the site of blood vessel injury. Proaggregatory and procoagulant platelets represent two distinct populations of activated platelets. While proaggregatory platelets facilitate the cessation of bleeding, or "hemostasis," by forming a plug of platelets clumped together through fibrinogen bridges, procoagulant platelets dramatically accelerate the coagulation cascade, culminating in fibrin clot formation. An interesting aspect of procoagulant platelets is that their morphology exhibits certain features of "necrosis" and "apoptosis." They may thus represent a form of cell death in platelets, albeit with an important role in thrombosis and hemostasis. This article introduces the concept of procoagulant platelets, their relevance to health and disease, and a comparison of existing methods for their analysis. It then provides comprehensive protocols for analyzing procoagulant platelets, investigating the mechanisms of their formation, and assessing their prothrombotic role in facilitating coagulation. The article concludes with a discussion of key steps, limitations, and troubleshooting principles for the described methods.

Introduction

There are at least two distinct populations of activated platelets^1,2. Pro-aggregatory platelets are characterized by high integrin activation and low, if any, PS exposure. On the other hand, procoagulant platelets are characterized by low integrin activity and high PS exposure^1,2, providing a surface for the assembly of tenase and prothrombinase complexes³, which is 10⁵-10⁶-fold and 300000-fold more active than individual soluble phase factor IXa and Xa, respectively⁴. Procoagulant platelets thus dramatically accelerate coagulation. An interesting aspect of procoagulant platelets is that their morphology resembles certain features of "necrosis" such as microvesiculation, ballooning of the cell with cytoskeletal disruption, and loss of membrane integrity, as well as those of "apoptosis" such as loss of membrane phospholipid asymmetry with exposure of phosphatidylserine in the outer leaflet^5,6. In other words, procoagulant platelet formation may represent a form of cell death in platelets, albeit with an important physiological function in hemostasis.

Procoagulant platelets are significantly associated with thrombotic disorders. While most healthy individuals do not have circulating procoagulant platelets, ~30% of normal donor platelets adopt a procoagulant phenotype ex vivo following exposure to strong agonists such as thrombin and collagen⁷. Circulating procoagulant platelets have been reported in trauma, where their formation may reflect activation by histone H4^8,9. In most prothrombotic disorders, however, increased levels of procoagulant platelets are only detected following ex vivo stimulation¹⁰. For example, patients with acute stroke in whom >51.1% of their platelets were converted to procoagulant platelets (also known as COATed platelets) by collagen and thrombin had a hazard ratio of 10.72 for recurrent stroke within 30 days compared to patients with lesser procoagulant platelet formation¹¹. Similar results have been reported in patients with transient ischemic attacks and carotid atherosclerosis¹². In contrast, the bleeding disorder Scott syndrome results from a mutation of ANO6, which encodes the phospholipid scramblase TMEM-16F, leading to deficient platelet PS exposure¹³. Idiopathic bleeding disorders and intracranial bleeding may be associated with a decreased ability to generate procoagulant platelets¹⁴.

Hence, assessing procoagulant platelets is part of any analysis of platelet function not only during basic investigations into mechanisms of platelet activation and consequent thrombosis and hemostasis, but also during clinical analysis for risk of thrombosis or bleeding in patients during various pathological states. An International Society on Thrombosis and Haemostasis (ISTH) panel recommended the use of Annexin V binding and P-selectin expression by flow cytometry for distinguishing procoagulants from other platelet subpopulations¹⁵. The article also discusses the various methods that can be used to analyze procoagulant and apoptotic platelets but falls short of describing the processes in detail. These methods include detection of (1) platelet activation by PAC1/JonA or fibrinogen binding (flow cytometry); (2) alpha-granule secretion by P-selectin expression (flow cytometry); (3) PS exposure by Annexin V/lactadherin binding (flow cytometry); (4) loss of membrane integrity by GSAO labeling (flow cytometry); (5) morphological changes like ballooning (microscopy); (6) detection of caspase activation by caspase assay (immunoblotting/luminometry/flow cytometry) or degradation of cytoskeletal substrate gelsolin (immunoblotting); (7) loss of mitochondrial membrane potential by mitochondrial potential-sensitive dyes such as JC-1/Mitotracker (flow cytometry); (8) mitochondrial intrinsic apoptotic markers Bax, Bak and cytochrome c release (immunoblotting); (9) procoagulant function by thrombin generation assay and coagulation factor Xa/Va binding (flow cytometry, microscopy); (10) cytosolic and mitochondrial calcium rise by fluorescent calcium sensitive dyes (flow cytometry, fluorometry, microscopy).

The present study delves into comprehensive protocols for the analysis of procoagulant platelets as well as distinguishing them from proaggregatory and apoptotic platelets. Most procedures described rely on flow cytometry that has the advantages of (1) being readily available and easy to use, (2) requiring low sample volume, and (3) allowing simultaneous detection of multiple subpopulations of platelets (proaggregatory, procoagulant, and apoptotic)¹⁵. These flow cytometry-based protocols are supplemented with functional assays of procoagulant activity based on coagulation factor binding and clot-based thrombin generation assays.

Protocol

Human participants were recruited in the study for peripheral venous blood sampling after obtaining written informed consent, strictly following the recommendations and approval of the Institutional Review Board of Cleveland Clinic Lerner Research Institute, with all study methodologies conforming to the standards set by the Declaration of Helsinki. Healthy adult participants above 18 years of age were included, while those younger than 18 years, individuals with a recent history of thrombotic events in the past six months, those with a history of alcoholism or drug abuse, and participants who had used anti-platelet or anti-coagulant medications in the past four weeks were excluded. A detailed description of materials and reagents used in the protocols can be found in the Table of Materials.

1. Platelet preparation

1. Draw peripheral venous blood samples into ACD anticoagulant (1:9 v/v) from human participants after obtaining written informed consent, strictly following the recommendations approved by the Institutional Review Board.
2. Centrifuge the blood collected in ACD at 100 x g for 20 min at 22 °C to obtain platelet-rich plasma (PRP) as the supernatant.
3. Centrifuge the PRP at 800 x g for 7 min at 22 °C after adding 3 µM of PGE₁ and 2 mM of EDTA.
4. Discard the supernatant and resuspend the platelet pellet in buffer A (see Table of Materials) by gentle pipetting.
5. Centrifuge the platelets resuspended in buffer A at 800 x g for 7 min at 22 °C.
6. Resuspend the platelet pellet in buffer B (see Table of Materials) by gentle pipetting.
7. Adjust the final platelet count to 1 × 10⁷/mL using an automated cell counter.
   NOTE: Perform all steps under sterile conditions and take precautions to maintain the platelets in a resting state by avoiding exposure to excessive shear during pipetting.

2. Analysis of procoagulant platelets by flow cytometry

1. Supplement washed human platelets with 2.5 mM calcium (0.5 µL of stock CaCl₂ 0.5 M solution in 100 µL of platelet suspension).
2. Keep one fraction of platelets unstimulated and stimulate the remaining fractions with thrombin (0.1 U/mL, 0.25 U/mL, 0.5 U/mL), convulxin (20 ng/mL, 50 ng/mL, 100 ng/mL), or their combination for 15 min at room temperature.
3. After stimulation, add 1 µL each of PE-Annexin V, FITC-PAC1, and APC-anti-human CD62P antibody to 100 µL of stimulated platelet suspensions.
4. Incubate the platelets in the dark at room temperature for 30 min.
5. Fix the platelets by adding an equal volume (1:1) of 2% formalin.
6. Analyze the samples by flow cytometry to quantify procoagulant platelets^15,16.
7. Draw an amorphous gate to encompass platelets, separating them from noise and multi-platelet particles.
8. Collect all fluorescence data using four-quadrant logarithmic amplification for 10,000 events in the platelet gate from each sample.
9. Define regions for platelets based on fluorescence positivity or negativity for phosphatidylserine (PS) exposure (Annexin V binding), P-selectin (CD62P) expression, and integrin αIIbβ₃ activation (PAC1 binding).
10. Consider platelets positive for both PS exposure (Annexin V binding) and P-selectin (CD62P) expression but negative for integrin αIIbβ₃ activation (PAC1 binding) as procoagulant platelets (Figure 1) (Table 1).
11. Similarly, consider platelets positive for both integrin αIIbβ₃ activation (PAC1 binding) and P-selectin (CD62P) expression but negative for PS exposure (Annexin V binding) as proaggregatory platelets.
    NOTE: Platelets negative for both integrin αIIbβ₃ activation (PAC1 binding) and P-selectin (CD62P) expression but positive for PS exposure (Annexin V binding) are likely apoptotic platelets^15,16.

3. Analysis of mitochondrial calcium by flow cytometry

1. Dilute washed human platelets to 1 × 10⁶/mL and supplement with 2.5 mM calcium (0.5 µL of stock CaCl₂ 0.5 M solution in 100 µL platelet suspension).
2. Label the platelets with 5 µM Rhod-2 AM (for mitochondrial calcium) for 30 min in the dark.
3. Gate platelets appropriately, as described in step 2.
4. Analyze events in the platelet gate for time-dependent changes in the mean fluorescence of acquired events over 5 min using a density scatter plot of PE fluorescence (for Rhod-2 AM) versus time.
5. Record baseline calcium levels for the first 1 min.
6. Add thrombin (0.1 U/mL, 0.25 U/mL, or 0.5 U/mL), convulxin (20 ng/mL, 50 ng/mL, or 100 ng/mL), or their combination, and continue acquiring data for another 4 min¹⁷.

4. Analysis of mitochondrial membrane potential by flow cytometry

1. Supplement washed human platelets with 2.5 mM calcium (0.5 µL of stock CaCl₂ 0.5 M solution in 100 µL of platelet suspension).
2. Keep one fraction of platelets unstimulated and stimulate the remaining fractions with thrombin (0.1 U/mL, 0.25 U/mL, or 0.5 U/mL), convulxin (20 ng/mL, 50 ng/mL, or 100 ng/mL), or their combination for 15 min at room temperature.
3. After stimulation, add mitochondria labeling dye (see Table of Materials) at a final concentration of 500 nM to the stimulated platelet suspensions.
4. Incubate the platelets for 30 min in the dark.
5. Fix the platelets by adding an equal volume of 2% formalin.
6. Analyze samples by flow cytometry¹⁷.
7. Gate platelets appropriately as described in step 2.
8. Analyze events in the platelet gate for a drop in fluorescence in the PE channel (for the mitochondria labelling dye) of the flow cytometer.

5. Analysis of caspase 3 and caspase 8 activity by flow cytometry

1. Supplement washed human platelets with 2.5 mM calcium (0.5 µL of stock CaCl₂ 0.5 M solution in 100 µL of platelet suspension).
2. Keep one fraction of platelets unstimulated and stimulate the remaining fractions with thrombin (0.1 U/mL, 0.25 U/mL, or 0.5 U/mL), convulxin (20 ng/mL, 50 ng/mL, or 100 ng/mL), or their combination for 15 min at room temperature.
3. After stimulation, add either 1:1000 (v/v) apoptosis detection reagent (see Table of Materials) (for both intrinsic and extrinsic apoptosis pathways) or 1:300 (v/v) FITC-IETD-FMK (for caspase 8 of the extrinsic apoptosis pathway) to the stimulated platelet suspensions.
4. Incubate the platelets for 30 min in the dark.
5. Fix the platelets by adding an equal volume of 2% formalin.
6. Analyze samples by flow cytometry¹⁷.
7. Gate platelets appropriately, as described in step 2.
8. Analyze events in the platelet gate for fluorescence in the FITC channel (for either caspase 3/7 or caspase 8) of the flow cytometer.

6. Analysis of prothrombin binding by flow cytometry

1. Conjugate bovine prothrombin with Alexa Fluor 488 dye using a protein labeling kit (see Table of Materials), following the manufacturer's instructions.
2. Supplement washed human platelets with 2.5 mM calcium (0.5 µL of stock CaCl₂ 0.5 M solution in 100 µL platelet suspension).
3. Keep one fraction of platelets unstimulated and stimulate the remaining fractions with thrombin (0.1 U/mL, 0.25 U/mL, or 0.5 U/mL), convulxin (20 ng/mL, 50 ng/mL, or 100 ng/mL), or their combination for 15 min at room temperature.
4. After stimulation, add AF488-conjugated bovine prothrombin (100 µg/mL) and 1 µL each of PE-Annexin V and APC anti-human CD62P antibody to 100 µL of stimulated platelet suspensions.
5. Fix the platelets by adding an equal volume of 2% formalin.
6. Analyze samples by flow cytometry¹⁷ for the quantification of prothrombin binding to platelets.
7. Draw an amorphous gate to encompass platelets separate from noise and multi-platelet particles.
8. Collect all fluorescence data using four-quadrant logarithmic amplification for 10,000 events in the platelet gate from each sample.
9. Draw regions for platelets with fluorescence positive or negative for PS exposure (Annexin V binding), P-selectin (CD62P) expression, and AF488-prothrombin (prothrombin binding).
10. Determine the proportion of all platelet events, as well as the proportion of platelets positive for both PS exposure (Annexin V binding) and P-selectin (CD62P) expression (procoagulant platelets), that are positive for prothrombin binding.

7. Analysis of prothrombin binding by confocal microscopy

1. Conjugate bovine prothrombin with Alexa Fluor 488 dye using a protein labeling kit (see Table of Materials), following the manufacturer's instructions.
2. Supplement washed human platelets with 2.5 mM calcium (0.5 µL of stock CaCl₂ 0.5 M solution in 100 µL platelet suspension).
3. Keep one fraction of platelets unstimulated and stimulate the remaining fractions with thrombin (0.1 U/mL, 0.25 U/mL, or 0.5 U/mL), convulxin (20 ng/mL, 50 ng/mL, or 100 ng/mL), or their combination for 15 min at room temperature.
4. After stimulation, add AF488-conjugated bovine prothrombin (100 µg/mL) and 1 µL each of PE-Annexin V and APC anti-human CD62P antibody to 100 µL of stimulated platelet suspensions.
5. Fix the platelets by adding an equal volume of 2% formalin.
6. Pellet the fixed platelets onto poly-D-lysine-coated coverslips.
7. Mount coverslips on microscopy slides using an anti-fade solution.
8. Alternatively, coat coverslips with collagen (100 µg/mL) for 1 h in a humid chamber.
9. Block the coated coverslips with 0.5% BSA in PBS for 1 h.
10. Label the platelets with AF488-conjugated bovine prothrombin (100 µg/mL) and 1 µL each of PE-Annexin V and APC anti-human CD62P antibody.
11. Allow labeled platelets to adhere for 20 min on collagen-coated coverslips.
12. Wash coverslips with adherent platelets three times with PBS.
13. Fix coverslips with 2% paraformaldehyde for 1 h.
14. Mount coverslips on microscopy slides using an anti-fade solution.
15. Observe the slides under a confocal microscope at 63× objective magnification.
16. Analyze the images using Fiji software for fluorescence quantification.

8. Clot-based platelet phospholipid-dependent thrombin generation assay

1. Supplement washed human platelets with 2.5 mM calcium (0.5 µL of stock CaCl₂ 0.5 M solution in 100 µL of platelet suspension).
2. Keep one fraction of platelets unstimulated and stimulate the remaining fractions with thrombin (0.1 U/mL, 0.25 U/mL, or 0.5 U/mL), convulxin (20 ng/mL, 50 ng/mL, or 100 ng/mL), or their combination for 15 min at room temperature.
3. Add 100 µL of treated platelets to a mixture of 50 µL of pooled normal plasma and 50 µL of low-turbidity kaolin solution (20 mg/mL), preincubated at 37 °C for 5 min.
4. Add 5 mM of CaCl₂ to the mixture.
5. Monitor the clot formation by turbidimetry on a microplate reader, measuring absorbance at 660 nm every 60 s for 1 h.

Results

A proportion of activated platelets turn "procoagulant" with a characteristic increase in surface expression of both phosphatidylserine (PS) and P-selectin, which distinguishes them from "apoptotic" platelets that are positive only for PS exposure as well as "proaggregatory" platelets that are positive for P-selectin expression. We found that thrombin induces a dose-dependent increase in the proportion of procoagulant platelets positive for both P-selectin and PS expression as detected by binding ...

Discussion

Procoagulant platelets demonstrate marked and sustained increases in intracellular calcium upon stimulation²⁶, but may be derived through different mechanisms. They are generated upon strong agonist stimulation with collagen and thrombin through distinct mediators, including most prominently mitochondrial calcium influx^18,19 along the electrochemical gradient across the inner mitochondrial membrane upon agonist-induced cytosolic calcium el...

Materials

Name	Company	Catalog Number	Comments
Acid Citrate Dextrose (ACD) solution (For 1000 mL)			Tri- Sodium Citrate-            22 g Citric Acid-                            8 g Dextrose-                              24.5 g Water-                                   Make up volume to 1000 mL
Alexa Fluor 488 protein labelling kit	Invitrogen	A10235
APC Mouse Anti-Human CD62P	BD Pharmingen	550888
Bovine Prothrombin	Prolytix	BCP-1010
Buffer A (Platelet Preparation)			M.W         Conc. in 1X      For 100 mL 10X solution HEPES        238.30         20 mM                    4.766 g NaCl              58.44       134 mM                   7.83 g KCl                74.55        2.9 mM               216.19 mg MgCl2          203.30           1 mM              203.30 mg NaH2PO4    156.01        0.34 mM                53.04 mg NaHCO3        84 01           12 mM            1.01 g Water to 100 mL after adjusting pH to 6.2 Dilute 10X solution 1:10 (v/v) with Milli Q water just before platelet preparation to obtain the 1X solution that needs to be supplemented with the following                  Conc. in 1X      For 1 ml 1X solution EGTA           1 mM       Add 10 μL (1:100 v/v) 100 mM EGTA  Glucose         5 mM      Add 5 μL (1:200 v/v) 1 M glucose PGE1            3 μM      Add 3 μL (1:333 v/v) 1 mM PGE1 solution
Buffer B (Platelet Preparation)			M.W         Conc. in 1X      For 100 mL 10X solution HEPES        238.30         20 mM                    4.766 g NaCl              58.44       134 mM                   7.83 g KCl                74.55        2.9 mM               216.19 mg MgCl2          203.30           1 mM              203.30 mg NaH2PO4    156.01        0.34 mM                53.04 mg NaHCO3        84 01           12 mM            1.01 g Water to 100 mL after adjusting pH to 7.4 Dilute 10X solution 1:10 (v/v) with Milli Q water just before platelet preparation to obtain the 1X solution that needs to be supplemented with the following                  Conc. in 1X      For 1 ml 1X solution Glucose         5 mM      Add 5 μL (1:200 v/v) 1 M glucose
CaCl2 (0.5 M) (For 50ml)			Molecular weight of CaCl2.2H2O = 147.02 Dissolve 3.675 g of CaCl2.2H2O in 50 ml Milli Q water
CellEvent Caspase-3/7 Detection Reagents Green	Invitrogen	C10423	Apoptosis detection reagent
Convulxin	Enzo Life Sciences	ALX-350-100
EDTA (0.5 M; pH 8) (For 100 mL)			Molecular Weight of EDTA Na2.2H2 O: 372.24g Weigh 18.612 g and suspend in 50 ml Milli Q water and check the pH (pH~4) Slowly add 10N NaOH with stirring and monitor the pH. EDTA starts solubilizing at around pH 7 and is completely soluble at pH 8. Make up the volume to 100 ml with Milli Q water.
EGTA (100 mM; pH 7.4) (For 100 mL)			Molecular Weight: 380.4 Add 3.804 g EGTA in 50 ml Milli Q water and check the pH (pH~3) Add 10 N NaOH dropwise while stirring and monitor the pH EGTA becomes soluble at pH 7.0 (approx) Adjust pH to 7.4 Make up the volume to 100 ml with Milli Q water
FITC Mouse Anti-Human PAC-1	BD	340507
FITC-IETD-FMK Caspase 8 (active) staining kit	Abcam	ab65614
Mitotracker Red CMXRos (mitochindria labeling dye)	Invitrogen	M7512	Stock= 1 mM (50 µg dissolved in 90 µl DMSO) Sub-stock= 100 µM (10 µl Stock + 90 µl DMSO) Working concentration= 500nM (0.5 µl in 100 µl)
PE Annexin V	BD Pharmingen	560930
Prostaglandin E1	Sigma	P5515	Stock= 20 mM (1 mg dissolved in 141 µL DMSO) Sub-stock= 1 mM (10 µl Stock + 190 µL DMSO) Working concentration= 3 µM (3 µL in 1 mL)
Rhod-2 AM	Invitrogen	R1244	Stock= 5 mM (1 mg dissolved in 178 µL DMSO) Sub-stock= 100 µM (10 µL Stock + 90 µL DMSO) Working concentration= 500 nM (0.5 µL in 100 µL)
Thrombin from human plasma	Sigma	T7572