Platelets and Hemostasis1

1. All of the following are the normal hemostasic response to vascular injury, except:

3. Palpable purpura is seen in all except:

7. In Thrombotic Thrombocytopenic Purpura (TTP) which of the following is true?

8. Falsely low platelets (pseudothrombocytopenia) is seen in all except:

11. Bernard – Soulier syndrome is characterized by all except:

19. A falsely low platelet count can be seen in all of the following, except:

20. Macrothrombocytopenia is characteristically seen in all, except:

22. Thrombocytopenia is seen in all except:

23. Immune destruction of platelets is seen in all, except:

24. All of the following can be seen in TTP, except:

26. All of the following are used in the treatment of TTP, except:

The hemostatic system is a complex mosaic of activating or inhibitory feedback or feed-forward pathways, integrating its five major components.

Endomitosis is the characteristic feature of megakaryocyte development in which the DNA is repeatedly replicated in absence of nuclear or cytoplasmic division. Megakaryocyte is the cell where endomitosis begins. Thrombopoietin being the primary regulator of megakarocyte maturation is mainly produced by liver, though some amount is also produced by kidney, marrow stroma and skeletal muscle. The circulatory life span of platelet is approximately 10 days with normal platelet count, but shorter in patients with moderate (7 days) to severe (5 days) thrombocytopenia. Mature platelets are formed as proplatelet from megakaryocytes. Nucleus is not extruded from megakaryocyte.

Purpura can be of 2 types

The increment in platelet count depends on many factors but if all other factors are absent then 1 unit of whole blood derived random platelet increases the platelet count by 5,000/mm³ per square metre. So in a person of BSA 2 m² it should raise by 10000/mm³ and of 1.5 m² by 7500/mm³ respectively.

Thrombocytopenia is the key laboratory finding in HIT and ranges from 20 to 100 thousand/mm³ or 50–70% decline from baseline. For confirming diagnosis two assay prototypes are available. One is ELISA based for detection of Ig antibodies to heparin–PF4 complexes and the other measure heparin–dependent antibodies that activate platelets. Activation assays are not available commercially because specific platelet donors are needed each time and donor platelets vary greatly in their sensitivity to activation by HIT sera. 14C–serotonin release assay is the best established activation assay. As compared to antigen assay, activation assays have greater specificity. The risk status for HIT can be assessed by 4Ts.

Interpretation: 6-8 > high risk, 4-5 intermediate risk, 0-3 low risk

ITP is the most common cause of acquired severe thrombocytopenia and 50% of ITP cases are children, i.e. < 10 years of age.

In TTP PT/APTT and fibrinogen levels are usually normal or only mild perturbed. Mild elevations in fibrin degradation products occur in 50% of patients.

Pseudothrombocytopenia has been reported in association with the use of EDTA as an anticoagulant, with platelet cold agglutinins associated with multiple myeloma, platelet satellitism, giant platelets and platelet agglutination. Other anticoagulants such as sodium citrate, oxalate, acid citrate dextrose and heparin can also cause platelet agglutination but is less frequent.

IVIg, anti-D and steroids are the first line treatment options given in ITP. Rituximab is usually given in cases of chronic ITP who fail to steroids.

The typical patient is a multiparous, middle aged or elderly female who presents with the acute onset of bleeding due to severe thrombocytopenia, a mean of 6–8 days (range 1–14 days) after receiving a blood product containing platelet material. Sera of more than 90% of patients contain antibodies to HPA-1a which is expressed on 97–98% of Caucasians. Bleeding usually lasts for 10 days, but can persist for 60–120 days with fatality rates of 10%. The treatment of choice is IVIg 1g/kg/day × 2 days. More than 90% of patients respond within 2-3 days. HLA matched platelets are generally not useful.

Thrombocytopenia is present in nearly all patients and ranges from 20,000 to near–normal levels. Platelets are large and small. The hallmark of BSS is the failure of platelets to aggregate in response to ristocetin or botrocetin. BSS platelets are deficient in GPIb, GPIX and GPV.

Approximately 1/3rd of patients have symptoms of hemolytic anemia (due to microangiopathic hemolytic anemia). Hemolysis is indicated11 by an elevated reticulocyte count, serum LDH and decreased serum haptoglobin. DCT is almost always negative.

VWF attaches platelets to the endothelium and vessel walls and not the platelets to each other. The most potent form of VWF is the large multimeric form. The Endothelium secretes VWF by one of the two pathways. The constitutive pathway–directly coupled to VWF synthesis and occurs without stimulation and the regulated pathway–involving VWF stored in Weibel–Palade bodies, is initiated by the action of secretagogues. VWF secreted by the constitutive pathway is in the largest multimeric form. The constitutive secretion of VWF from endothelium maintains the Plasma VWF level. During activation after tissue injury, it is released from the Weibel–Palade bodies of and α-granules of platelet. VWF carries factor VIII and not IX.

The inhibitory pathways in platelets are CD39 (Ecto-ADPase), nitric oxide and PGE2 (at high concentrations) and PGI2 also called prostacyclins (at low concentrations)

The formation of a factor VIIa/TF complex at the site of injury is the major initiating event in hemostasis in vivo. The factor VIIa/TF complex can activate both factor IX and X. TF is normally expressed on pericytes around blood vessel and by epidermal, stromal and glial cells. In Contrast to other proenzymes, factor VII circulates in the blood in two forms: the inactive zymogen factor VII and the active form VIIa. The concentration of factor VIIa is low but sufficient to generate significant factor X–with the help of tissue factor.

The risk factor for DIC are infections, purpura fulminans, solid tumors, leukemias, trauma, brain injury, burns, liver disease, heat stroke, snake bites, hemangiomas, aortic aneurysm, transfusion reaction, abruptio placentae, amniotic fluid embolism, pre-eclampsia and eclampsia, HELLP syndrome, dead fetus syndrome, acute fatty liver of pregnancy.

Fibriongen levels are actually decreased and not increased in DIC.

Pseudothrombocytopenia is seen with giant platelets (macrothrombocytopenia) which is seen most commonly in West Bengal, India. In about 1% of individuals, EDTA causes platelet clumping, resulting in pseudothrombocytopenia. Occasionally platelets may be seen adhering to neutrophils (platelet satellitism). Presence of platelet cold agglutinins is seen with multiple myeloma. GP IIb/IIIa antagonists (e.g. abciximab) is associated with both pseudothrombocytopenia and true thrombocytopenia. Antiphospholipid antibodies are also associated with pseudothrombocytopenia.

May–Hegglin anomaly, Fechtner syndrome, Sebastian syndrome and Epstein syndrome are autosomal dominant macrothrombocytopenia with mutations in the MYH9 gene. Wiskott – Aldrich Syndrome is a rare X-linked immunodeficiency disorder characterized by microthrombocytopenia, eczema, recurrent infections, T-cell deficiency and increased risk for autoimmune and lympho proliferative disorders. Platelets appear larger than normal, pale ghost like, oval forms on blood smear in Gray–platelet syndrome. Thrombocytopenia is common and can be moderately severe. In Bernard-Soulier syndrome, thrombocytopenia is present in nearly all patients, but is variable in its severity, ranging from approximately 20,000/mm³ to normal levels.

Wiskott–Aldrich syndrome (WAS) is a rare X–linked immunodeficiency disorder characterized by microthrombocytopenia, eczema, recurrent infections, T-Cell deficiency and increased risk for autoimmune and lymphoproliferative disorders. The most consistent feature is microthrombocytopenia. The bleeding time is usually prolonged. There is variable abnormality in platelet aggregation and release of dense body contents.

Table on p. 1892 William's 8th ed

Henoch–Schönlein purpura is a pediatric vasculitic syndrome characterized by the acute onset of abdominal pain and lower extremity eruption of diffuse urticarial plaques and palpable purpura. It affects patients of 2 to 20 years of age. Several environmental triggers are present, e.g. viral (URI, Hep B, Hep C, HIV, parovirus B19) and bacterial (Streptococcus sp., S. aureus and Salmonella sp.). Adult disease is precipitated by NSAIDs, ACE inhibitors and antibiotics, food allergies and insect bites. Inspite of its chronic relapsing pattern, the long term evolution is benign in majority of patients. Glucocorticoids are reserved for cases with renal involvement.

Secondary cause of immune destruction of platelets is seen in:

TTP has manifestations of microangiopathic hemolytic anemia and thrombocytopenia. Many patients have fever. Thrombocytopenia typically is severe. Almost all patients have normal fibrinogen levels, PT and APTT.

The mainstay of therapy for TTP is plasma exchange with the exception of factor H deficiency and possibly APS syndrome and quinine–induced disease. No compelling evidence indicates that plasma therapy is effective for thrombotic micro-angiopathy caused by a mechanism other than ADAMTS 13 deficiency.

The treatment options for TTP are

This patient is a case of neonatal alloimmune thrombocytopenia (NAIT) so maternal serum will show anti–platelet antibodies which is most commonly against anti–HPA-1a antigen (78%) in whites. However in Asians most common is HPA-4a (80%). It is very important to differentiate between maternal ITP and NAIT and the features are

NAIT is like Rh hemolytic disease of newborn but the first born child is affected in 40-60% of cases (which is spared in Rh hemolytic disease of newborn). Platelet count recovers to normal in 1–2 weeks. The diagnosis is usually confirmed by tests for circulating maternal alloantibodies against fetal antigens (usually by MAIPA) or by platelet typing of the parents and neonate by genotyping or ELISA.

Postnatal management: Options are IVIg, glucocorticoids and platelet transfusions. In severe bleeding, platelets can be transfused and should be ABO and (Rh) D compatible and HPA-1a negative.

Prenatal management: In high-risk NAIT weekly IVIg/administration with or without glucocorticoids. In severe thrombocytopenia fetuses, early delivery with caesarean Section may reduce ICH.

This pregnant lady is most likely to have weak Rh D expression and should be labeled as Rh (D) positive which was missed in previous testing. Blood donors and pregnant women who type D negative using standard typing sera should be tested further for weak expression using more sensitive methods, such as an ICT. Donors with weak D antigen are considered positive. Testing for weak D is optional for transfusion recipients.

The alternatives are IVIg, steroids and platelet transfusion. IVIg and/or glucocorticoids therapy may increase platelet count rapidly. Platelet transfusion will not increase the platelet count rapidly in cases of NAIT15 unless it is HPA-1a negative which may not be available on urgent basis. IVIg acts faster than steroids so the best answer would be IVIg.

The differential diagnosis in this case are: with points in favor and against

Complete response occurs after an average of 9–16 days of plasma exchange, but approximately 25-50% of patients develop acute exacerbations within 2 weeks that requires plasma exchange. Usually seen in up to 1/3rd of patients, relapses are defined as recurrence occurring 30 days after a complete response occur. Most relapses occur during 1st year. Relapsing patients typically respond to plasma exchange therapy. Relapses in TTP are associated with severe ADAMTS 13 deficiency and detectable ADAMTS 13 autoantibody inhibitors. Immunosuppressive therapies mentioned in the explaination for Q. no. 26 are used only when the disease is refractory to plasma exchange therapy. Splenectomy can result in lasting remissions or reduce the frequency of relapses for some patients with TTP who are refractory to plasma exchange or immune suppressive therapy, presumably by removing a major site of antiADAMTS 13 antibody production.

16What was previously described as HIV–associated ITP is changed to primary HIV–associated thrombocytopenia (PHAT). PHAT is the most common cause of thrombocytopenia in persons with HIV. Differentiating features of PHAT from denovo ITP are

HAART is an important treatment modality in patients with PHAT and should be the initial treatment of choice. There is no history of any drug exposure besides LMWH and compared to UFH there is less risk of HIT. The most likely cause of thrombocytopenia in this case is ITP so the treatment of choice is high dose IVIg if the patient is bleeding. As the patient has relapsed and not on HAART, so it needs to be started. Obviously the LMWH needs to be stopped but no anticoagulation should be given in view of low platelet count.