A Practical Guide to Haemostasis

Platelet Function Testing:
Platelet Procoagulant Activity [PCA] Assays

Scott Syndrome and Platelet Procoagulant Activity

The procoagulant activity of platelets has previously been designated Platelet Factor-3 (PF3) or Platelet Factor-3 availability (PF3a).  Scott syndrome is named after the first patient described with this disorder.

Scott syndrome is a rare disorder of platelet procoagulant activity which appears to be due to mutations within the ANO6 gene [previously termed TMEM16F] located on the long arm of chromosome 12 at 12q12 and which encodes ANO6 scramblase and which is involved in the movement of Phospholipid [Phosphatidylserine [PS]] - from the inner leaflet of the platelet membrane to the outer leaflet - also known as the 'flip-flop' mechanism.

Mutations within the ANO6 gene leads to an abnormal ANO6 scramblase and defective translocation of Phosphatidylserine [PS] - from the inner leaflet of the platelet membrane to the outer leaflet.  PS exposure on the surface of activated platelets serves as a scaffold for the assembly of the Tenase and Prothrombinase complexes and defects in this lead to impaired Thrombin generation .  Phosphatidylserine exposure in platelets is under the control of three types of proteins:
  - Flippases (inward directed pumps)
  - Floppases (outward directed pumps) and
  - Scramblases, which can generate bidirectional redistribution.
After platelet activation the Scramblase and Floppase are triggered whilst the Flippase is inhibited.

Scott syndrome patients suffer from a moderate-to-severe haemorrhagic disorder.  However, there are other disorders of platelet procoagulant activity including Quebec Platelet Disorder (Platelet Factor V Quebec), Platelet Factor V-New York, some patients with Storage Pool Disorder [SPD], Glanzmann's Thrombasthenia and Bernard Soulier Syndrome.  The findings in Glanzmann's Thrombasthenia and Bernard Soulier Syndrome suggest that the abnormalities in the platelet membrane glycoproteins may also interfere with platelet PCA and the bleeding tendency is not due entirely to lack of the relevant glycoprotein.

At least some of the patients with Scott Syndrome show impaired binding of Factor Va to activated platelets. The changes in the phospholipid membrane that occur with activation of the platelet are crucial for the formation of the ‘Tenase’ and ‘Prothrombinase’ complexes and hence the defective Va binding demonstrated by flow cytometry,


Several tests may be used to screen for abnormalities of platelet procoagulant activity:

1. Prothrombin Consumption Index [PCI]:
This is based on the finding that that the Prothrombin Time of serum is longer than that of plasma, as Prothrombin is normally consumed during its conversion to Thrombin.
          PCI = [Serum Clotting Time/Plasma Clotting Time] x 100%
If thrombocytopenia is excluded, then an abnormal PCI which is corrected by normal platelets or phospholipid raises the possibility of a defect in platelet PCA.

2. Platelet Factor 3 Availability (PF3a) Assay:
The PF3a assay relies on the principle that incubation of platelet-rich plasma (PRP) with kaolin activates the procoagulant activity of platelets, resulting in a progressive shortening of both the recalcification time and Russell Viper Venom time. By appropriate mixing of normal PPP and PRP from a patient, the PF3a test can be made specific for assessing the PCA of platelets
Abnormal PF3a has been reported in patients with both congenital and acquired bleeding disorders and may be associated with other defects in platelet function.
PF3a is a useful screening test for detecting an abnormality in platelet PCA but is not diagnostic of any specific disorder.

3. Annexin V binding:
Annexin V is a protein with a high affinity and specificity for aminophospholipids at physiological calcium concentrations and can be used as a marker of platelet activation and the development of procoagulant activity. Labelling PS with fluorochrome-conjugated Annexin V before and after platelet activation demonstrates the flip-flop effect of activation.  The test can be performed on whole blood by Flow Cytometry and provides a rapid method to assess procoagulant activity in platelets.

What Test Next

If the Prothrombin Consumption Index [PCI] or the Platelet Factor 3 Availability (PF3a) assay suggests an abnormality - then the cause of this should be sought. A screen for mutations within the ANO6 gene should be undertaken but also consideration of the other causes of an abnormal PCA screen.

In many cases PCA assays are not readily available and in such cases, mutational analysis of the genes involved in platelet production and function is a logical approach to investigating a case of an unknown bleeding disorder.