Methods
Patients and blood collection
We enrolled patients who received treatment in Dmitry Rogachev National
Medical Research Center to this study. The key inclusion criteria were:
the presence of a vascular tumor (kaposiform hemangioendothelioma or
bundle angioma) and associated coagulopathy and thrombocytopenia at
diagnosis. Patients, who received platelet transfusions within 3 weeks
prior to enrolment, were excluded from the study. Age and treatment
history were not exclusion criteria. Age-matched healthy volunteers were
recruited as controls to this study.
At the enrollment, 1.6 mL of blood were collected into hirudin
containing S-Monovette® tubes, and 4.3 mL of blood were collected into
sodium citrate containing S-Monovette® tubes (Monovette, Sarstedt,
Newton, NC). Blood samples were processed within 1.5 hours after
collection.
The study protocol was approved by the independent ethics committee of
the CTP PCP RAS (approval number 3/1-21, 05.10.2021). All participants
provided written informed consent before enrolment. The study was
conducted in accordance with the principles of the Declaration of
Helsinki and the International Conference on Harmonization Good Clinical
Practice Guideline. Data available on request from the authors.
Materials
The materials were as follows: calcium-sensitive cell-permeable
fluorescent dye Fura-Red-AM, (Molecular Probes, Eugene, USA); ADP, EGTA,
HEPES, bovine serum albumin, apyrase grade VII, TRAP (SFLLRN), AYGPKF,
mepacrine (Sigma-Aldrich, St Louis, USA); CD62p-Alexa647, CD42b-PE,
CD61-Alexa647, PAC1-FITC (Sony Biotechnology, San Jose, USA), collagen
(NPO Renam, Moscow, Russia). Cysteine-containing version of cross-linked
collagen-related peptide (CRP) was kindly provided by Prof. R.W.
Farndale (the University of Cambridge, Cambridge, UK). Rhodocytin was
isolated as previously described 23,24(p). CLEC-2
inhibitors 2CP 25 and HB125 were synthesized as
described in Supporting information.
Tyrode’s buffer (150 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 2
mM CaCl2, 0.4 mM
NaH2PO4, 0.4 mM
Na2CO3, 5 mM HEPES, 5 mM glucose, 0.5%
BSA, pH 7.35) was freshly prepared from reagents (Sigma-Aldrich, St
Louis, MO).
Endpoint flow cytometry
Functional testing of the platelets was performed as described26. Briefly, blood was collected into sodium citrate
(3.8% v/v) vacuum tubes and then diluted 20 times by Tyrode’s buffer
without 2.5 mM calcium. Samples were then incubated 1:1 with activators
(12.5 µM SFLLRN and 10 µg/mL CRP) or (12.5 µM SFLLRN, 200 µM AYGPKF and
2 µM ADP) or vehicle in Tyrode’s-Calcium (5 mM) buffer. After 10 min
incubation, samples were mixed 1:1 with anti-CD42b, anti-CD61, PAC1,
anti-CD62p, and Annexin-V in Tyrode’s-Calcium (2.5 mM) buffer and left
for 10 minutes. Alternatively, platelets were incubated with 10 µM
mepacrine for 30 minutes. After this, samples were diluted to the
platelet concentration of 5000 plt/µL by Tyrode’s Calcium (2.5 mM)
buffer and studied using NovoCyte Acea Flow cytometer.
Continuous flow cytometry
Assays of platelet signaling were performed as described27. Hirudinated whole blood was incubated with 2 µM of
Fura-Red in the presence of 1 U/mL of apyrase for 35 minutes at
37oC. Leukocyte rich plasma (LRP) was collected,
diluted by the Tyrode’s-Calcium buffer to the final concentration of
1000 plt/µL, and left resting for 30 minutes. 100 µL was the lowest LRP
volume used in this study. 100 µg/mL of Alexa-488 labeled human
fibrinogen was added 2 minutes before sample loading to the BD FACS
Canto II Flow Cytometer (BD Biosciences, San Jose, US). Samples were
analyzed in a continuous mode. The primary fluorescence signal was
averaged over equal periods. The ratio of calcium bound Fura-Red
(excited by 405 nm laser) to calcium-free Fura-Red (excited by 488 nm
laser) was recalculated into platelet cytosolic calcium concentration
using Grynkiewicz formula 28.
Laser-scanning
aggregometry
Low angle scattering analysis of platelet activation was performed as
described elsewhere 29. Briefly, LaSca method is based
on the measurement of low angle light scattering in a diluted platelet
suspension. This method is used to characterize the platelets function,
allowing simultaneous recording of platelet di-aggregate formation
(light scattering at 1.5°) and shape changing (light scattering at 12°).
Platelet-rich plasma was diluted by Tyrode’s buffer to platelet
concentration 10 000 plt/μL, and the initial velocity and aggregation
amplitude upon platelet stimulation were evaluated.