The Effects of Shear Stress on Platelets Treated with Antibodies Against Mechanosensory Receptors

Overview

This video demonstrates the method to assess the impact of shear stress on platelets. Antibody treatment induces dissociation and conformational changes in mechanoreceptor complexes, activating the platelets and expressing markers on the surface. These markers are observed via immunostaining and flow cytometry.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Blood Draw and Platelet Isolation

1. Draw human blood from consenting healthy adult donors via venipuncture on the day of the experiment into 3.8% trisodium citrate. One 4.5 mL tube of blood is sufficient to yield enough platelet-rich plasma (PRP) for 20-25 conditions in donors whose platelet counts are close to 250 x 10³ per μL.   
   NOTE: Avoid drawing blood via narrow gauge needles (smaller than 21 G).
2. Prepare PRP via centrifugation at 22 °C and 140 x g for 12 min with a long brake. This will result in two distinct layers, with red blood cells at the bottom and the light-colored PRP at the top.
3. Isolate the top, cloudy, yellow layer of PRP via careful pipetting through a pipette tip cut at a 45° angle and obtain the platelet count via a complete blood count (CBC).
4. If necessary, wash platelets in 1,4-Piperazinediethanesulfonic acid sodium salt (PIPES)-buffered saline (150 mM sodium chloride, NaCl, 20 mM PIPES) in the presence of prostaglandin E1 (PGE1), and resuspend in Tyrode's buffer (134 mM NaCl, 0.34 mM di-sodium hydrogen phosphate, Na₂HPO₄, 2.9 mM potassium chloride, KCl, 1 mM magnesium chloride, MgCl₂, 5 mM glucose, 12 mM sodium bicarbonate, NaHCO₃, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, HEPES, pH 7.35) with 5 mM glucose, otherwise, proceed to step 1.5. The following steps describe the washing in brief.
   1. Adjust PRP volume to 10 mL with PIPES-buffered saline and add 0.6 μM PGE1.
   2. Centrifuge for 8 min at 1,900 x g, then discard the supernatant and let the platelet pellet sit in 400 μL of Tyrode's and glucose solution for 5 min.
   3. Gently resuspend the platelet pellet and keep it undisturbed for 30 min.
5. Adjust the platelet count to ~250 x 10³ platelets per μL with pooled human platelet-poor plasma (PPP) and maintain the suspension at 22 °C undisturbed or under gentle rotation.

2. Antibody and uniform shear treatment

NOTE: All steps in section 2 that require pipetting should be done slowly, so as not to introduce any shear.

1. Add the desired antibody to the PRP or washed platelets and mix gently by pipetting up and down or stirring with a pipette tip. Leave it undisturbed at room temperature for 5-10 min. Add an equivalent volume of PPP or Tyrode's buffer to a negative control.
2. Turn on the cone-plate viscometer, set the plate temperature to 22 °C, and allow time to let the plate reach this temperature.
3. Pipette the treated PRP or washed platelets onto the temperature-controlled cone-plate viscometer directly at the center of the plate. Ensure that all of the sample is deposited between the cone and plate at the point of contact, and not on the outside of the cone's rim.
4. Shear at an appropriate rate and duration.
   1. Calculate shear as indicated by the viscometer manual, or as previously shown.
   2. Determine shear rate from viscosity and desired shear stress via Newton's law of viscosity; ; plasma viscosity is 1.5-1.6 centipoise (cP). For example, a normal shear range for human circulation is 5-30 dyn/cm², and shear should be applied on the single-digit minute time scale.
5. Lift the cone off of the plate slightly (~2 mm) so that the sample remains in contact with both the plate and cone and use a gel-loading or other long pipette tip to collect 5-10 μL from the center of the sample volume.
6. Incubate the sheared samples with the desired markers for 20 min at room temperature. For markers of phosphatidylserine, β-galactose, and P-selectin exposure use Lactadherin C2 domain (LactC2) at 0.08 μM, Erythrina cristagalli lectin (ECL) at 6.25 μg/mL, and anti-P-selectin antibody (20 µg/mL), respectively.
7. Fix samples in 2% paraformaldehyde for 20 min at RT prior to dilution or cold storage and proceed to step 3.1 or store samples at 4 °C for no longer than 12 h.

3. Detection of surface markers and crosslinking via flow cytometry

1. Analyze the sample via flow cytometry, collecting at least 20,000 events for each condition.
2. Quantitate signal strength of the fluorescent markers using the height value for the intensity of each fluorophore, or the geometric mean fluorescence intensity (MFI).
3. If aiming to detect platelet crosslinking following shear treatment, analyze the sample on an imaging-capable flow cytometer and quantitate crosslinking by area and aspect ratio parameters.
   1. Plot a histogram of area and/or aspect ratio.
   2. Use a negative control with bovine serum albumin (BSA) or vehicle to draw a gate excluding most fully circular events (this gate is usually drawn at an aspect ratio ~0.8) and quantitate the percentage of events inside of this gate. Events with a lower aspect ratio are more likely to be cross-linked.

Materials

Name	Company	Catalog Number	Comments
APC anti-human CD62P (P-Selectin)	BioLegend	304910
Brookfield Cap 2000+ Viscometer	Brookfield	-
FITC-conjugated Erythrina cristagalli lectin (ECL)	Vector Labs	FL-1141
Pooled Normal Human Plasma	Precision Biologic	CCN-10
Vacutainer Light Blue Blood Collection Tube (Sodium Citrate)	BD	369714
Vacutainer Blood Collection Set, 21G x ¾" Needle	BD	367287