Disorders of HemostasisDr. Raymond L. Lobins

Cox health – Springfield, Missouri

Bleeding and Clotting are in Balance

Vasoconstriction

! 1. Vasoconstriction of a damaged blood vessel slows the flow of blood and thus helps to limit blood loss. This process is mediated by:

! Local controls. Vasoconstrictors such as thromboxane are released at the site of the injury.

! Systemic control. Epinephrine released by the adrenal glands stimulates general vasoconstriction.

Coagulation pathways Platelet Adhesion 1.Cross links collagen to platelets (PLT)

! vWF released from endothelium and floating in blood binds with collagen.

! Rolling PLT Bind to vWF via gpIb/V/IX receptor.

! PLT bound to vWF changes shape, releases granules (alpha and dense).

! PLT shape change also opens the gp IIb/IIIa receptor to allow binding with fibrinogen.

Platelet activation - Platelet Granules

! Alpha Granules – there are many types of alpha granules that contain varied products. But alpha granules do contain vWF, FV, and FXIII and fibrinogen!

! Dense granules - contain a range of small molecules such as ADP, ATP, GDP, serotonin, pyrophosphate, magnesium and calcium

! Release of granules supply many of the products needed to attract more platelets, maintain vaso-constriction, and start secondary hemostasis.

! ADP – activates cell via P2Y12 receptor, involved in Ca++ regulation.

! Platelets also secrete Thromboxane A2 – blocks prostacyclin (via gpVI). vasoconstriction and activation of IIb/IIIa receptor.

! Serotonin – vasoconstriction.

Platelet Aggregation

Disorders of HemostasisPrimary Hemostasis

Signs and Symptoms of defects in primary hemostasis

Primary hemostasis disorders

! Vascular anomalies.

! Thrombocytopenia.

! Von Willebrand disease.

! Platelet function disorders.

Vascular Anomalies! Structural anomalies - such as ! hereditary hemorrhagic telangiectasia.

! disorders of the connective tissue (including Ehlers–Danlos disease and osteogenesis imperfecta). – these are your “rubber men”

! small vessel vasculitis

! Generalized vascular fragility dominates the clinical picture.

! Can lead to severe varicosities and arterial rupture, which may cause sudden death (usually in the third or fourth decade of life).

Thrombocytopenia Von Willebrand disease

! Most common coagulopathy (1 -2% of population).

! Von Willebrand is stored in endothelial cells and platelets.

! Composed of repeating multimers.

! Most types are Autosomal dominant (types 2N and 3 are recessive).

! Normal plasma corrects the abnormal bleeding time.

! Ristocetin co-factor measures activity of vWF.

Alterations in vWF Concentration! INCREASED

! Pregnancy ! Physical Stress ! Epinephrine ! Emotional Stress ! Estrogens ! Thyroid hormones ! Exercise ! Elderly !Diabetes

! DECREASD

! Wilms tumor ! Type O blood type ! VSD ! Congenital Heart dz. ! Monoclonal

gammopathies ! Myelo/lympho-

proliferative disorders

Types of vWD

Von Willebrand disease

! 182kb produce a 2813 amino acid single peptide pro-VWF

! vWF is composed of repeating multimers of dimers 500kD-10,00kD.

! Requires cleaving for active vWF. ! Cleavage by ADAMTS13 @ A2

inactivate vWF

! Smaller multimers and subunits can bind to factor VIII at domains C & D

! Factor VIII/vWF complex protects Factor VIII from proteolysis and extends it’s half life.

! Location – Chromosome 12 at 12p13.2

! Pseudo-vWF location - chromosome 22

! Secreted in 2 ways (regulated & unregulated).

! Platelets secretion is only via regulated pathway…..therefore…..

! All circulating vWF is by endothelial excretion.

Von Willebrand disease screening tests

! Bleeding time – insensitive & nonspecific

! Platelet function analyzer-100 (PFA-100) 1996 2 types of cartridges (col/epi & col/adp). Blood passed thru cartridge at high pressures – time to clot is measured. Closure time is increased in primary hemostasis disorders. For type1 vWD col/epi – more sensitive & specific.

!ABO typing

! PTT – prolonged if VIII< 30% of normal

Von Willebrand disease specific tests

!vWF antigen – measures quantity. Some labs have separate references for type O.

!Ristocetin cofactor – measures activity of vWF ristocetin cleaves vWF allowing it to bind to gpIb.

!Collagen binding assay –measures how well plasma vWF binds collagen.

!Factor VIII level

Von Willebrand disease type specific tests

!Ristocetin induced platelet aggregation (RIPA)- To determine qualitative defect in vWF binding Looking for spontaneous binding or binding at low concentration (IIB or pseudo-vWD)

!vWF multimers- detected by gel electrophoresis. Can detect absence of lg multimers (IIa) or if larger than normal (Vicenza, TTP, neonates)

AlgorhythmPlatelet function disorders - Storage pool deficiency (SPD).

! Rare. ! Deficiencies in platelet secretion granules, changing the contents of

! dense granules (δ-SPD) = (ADP and Serotonin), alpha-granules (α-SPD or gray platelet syndrome), or both (αδ-SPD).

! Can be idiopathic or part of a complex disorder such as Hermansky–Pudlak syndrome, Chediak-Higashi syndrome, Wiskott-Aldrich syndrome, and thrombocytopenia-absent radius (TAR).

! Bleeding tendency is usually mild.

Platelet function disorders! Glanzmann thrombasthenia ! Rare.

Autosomal recessive.

! Defect in the platelet membrane receptor GPIIb-IIIa (the main fibrinogen receptor on the platelet surface). Ineffective platelet aggregation.

! Genetic predisposition (French Gypsies, Iraqi Jews, Jordanian Arabs, and South Indians).

! Normal platelet morphology and normal platelet count.

! Do not aggregate to ristocetin.

Platelet function disorders! Bernard–Soulier syndrome ! Rare.

Autosomal recessive.

! Defect in one of the components of the GP Ib-IX-V complex. Abnormally rapid removal of the bizarre platelets may be responsible for thrombocytopenia

! Noted by high phospholipid content in platelets. ! Giant platelets & thrombocytopenia. ! Do not aggregate to ristocetin.

Disorders of HemostasisSecondary Hemostasis

Secondary Coagulation – Plug Stability

Secondary Coagulation! Extrinsic pathway: ! Tissue factor (cofactor) and FVII (pro-

enzyme; FVIIa is the enzyme) + (Ca2+)

! The TF-FVIIa complex activates FX and also activate FIX

! Under physiologic conditions (blood vessel injury), FXa is generated by the TF-FVIIa complex on the surface of fibroblasts.

! Initiation of thrombin generation occurs through extrinsic pathway, which is expressed on fibroblasts.

! This generates small amounts of thrombin and Factor IXa – leads to thrombin burst.

! Intrinsic pathway: ! Enzymatic coagulation factors: ! FXII, FXI, and FIX and a cofactor

(non-enzyme): FVIII, along with Ca2+ .

! Occurs on the phospholipid surface(PS).

! The ultimate product of the intrinsic pathway is activated FIX (Factor Ixa).

! Thus both intrinsic & extrinsic pathways converge at the activation of FX.

! however the site of activation of FX differs (fibroblast for the extrinsic pathway and platelet for the intrinsic pathway).

Secondary Coagulation

! Common pathway: ! Enzymatic coagulation factors: FX and prothrombin (FII) with FV (a cofactor)

& Ca2+

! Works on a Phospholipid surface (PS) and results in fibrinogen to Fibrin.

! FXa, with the aid of its cofactor FVa, activates prothrombin to thrombin.

! Thrombin cleaves fibrinogen to soluble fibrin, which is then crosslinked to insoluble fibrin by FXIIIa (FXIII is also activated by thrombin).

Defects in Secondary Hemostasis

Defects in Secondary Hemostasis (Disorders of Fibrin Formation)

! Hereditary vs acquired ! Quantitative vs qualitative deficiencies

! Laboratory screening tests (PT, APTT) ! Does not differentiate quantitative vs qualitative disorders

! Qualitative abnormal proteins will ! Prolong clotting test

! Being recognized by immunologically-based procedures

! Activity assays ! Essential when screening for deficiencies

Disorders of Proteins of Fibrin Formation

Diagnostic testing – in General

! Isolated High PTT – think deficiency in factors VIII, IX, and / or XI.

! Isolated High PT – think deficiency in factor VII.

! Both PT and PTT high – think deficiency in factors II, V, or X.

! Both PT and PTT high and thrombin time increased – think fibrinogen deficiency.

! PT, PTT, thrombin time, and bleeding time all normal – think factor XIII.

○ Hemophilia A ● Factor VIII Deficiency - 1/5000

○ Antihemophilic Factor

○ X-linked recessive disorder

○ Most common type of hemophilia

○ Hemophilia B ● Factor IX Deficiency – 1/20,000

○ Christmas Factor (from family of first patients diagnosed with the disorder)

○ X-linked recessive disorder

○ Hemophilia C ● Factor XI Deficiency – 1/100,000

● Autosomal recessive disorder seen primarily in the Ashkenazi Jewish population

● Symptoms range from mild to severe

Hemophilia

! Insufficient generation of thrombin ! Due to lack of FIXa/VIIIa complex through the intrinsic pathway.

! Bleeding severity complicated by excessive fibrinolysis

! Clinical severity corresponds with level of factor activity

Hemophilia

! Severe hemophilia ! Factor coagulant activity <1% of normal

! Frequent spontaneous bleeding into joints and soft tissues

! Prolonged bleeding with trauma or surgery

! Moderate hemophilia ! Factor coagulant activity 1-5% of normal

! Occasional spontaneous bleeding

! Excessive bleeding with surgery or trauma

! Mild hemophilia ! Factor coagulant activity >5% of normal

! Usually no spontaneous bleeding

! Excessive bleeding with surgery or trauma

Hemophilia severities

! Replacement of clotting factor to achieve hemostasis ! Annual cost for patient with severe hemophilia

! $20,000-100,000

! Various products available ! Plasma-derived low, intermediate and high purity products ! Plasma-derived ultrapure products ! Ultrapure recombinant products

! Replacement products – benefits vs risks ! Blood-born pathogens

! Hepatitis A, B, C, G; HIV, Parvovirus B-19 ! Thrombotic complications with some F-IX concentrations ! Development of alloantibody inhibitors

! Neutralize coagulant effects of replacement therapy

Hemophilia – Treatment

Factor Replacement! Plasma derived (purified) –

Alphanate, Koate, Wilate, Humate-P. T1/2 = 12hr, thus treatment = 3x/week.

! Recombinant VIII – Made from DNA in animal cell lines. – (more immunogenic). Recombinate, Kogenate, Helixate, Advate. T1/2 = 12hr, thus treatment = 3x/week.

! Recombinant VIII Fc fusion protein – Eloctate. T1/2 = 18hr, thus treatment = q4-5 days.

! Recombinant IX - Benefix (T1/2 = 33hr), thus treatment = 2x/week.

! Recombinant IX Fc fusion protein or IX-FP (albumin). Alprolix (T1/2 = 82hr) or Idelvion (T1/2 = 102hr).

Treatment options - historically! Historically Prothrombin complex concentrates (PCC) 3F – 4F.

(FFP is not a concentrate).

! Enriched concentrates of factors II, IX, X, (VIIa – 4F, some with protein c and s).

! Named by there level of factor IX in them.

! Are not primarily used for hemophilia treatment any longer. Usually used for bleeding or anticoagulation reversal.

! Typically one dose (rarely may need more than one dose).

! Primary benefit over FFP = volume infused and time to correction (30minutes vs. up to 24hr).

! Of note: 4F-PCC did reverse “lab effects” in healthy donors from Rivaroxiban.

Replacement dosing

! Dose calculations –

! 30-40% for most mild hemorrhages. > 50% for severe bleeds (trauma / major dental surgery / major surgery). 80-100% in life-threatening hemorrhage.

! Hospitalization is reserved for severe or life-threatening bleeds, such as large-soft tissue bleeds; retroperitoneal hemorrhage or other internal bleeding; and hemorrhage related to head injury, surgery, or dental work.

Dosing principles – standard products

! Factor VIII – 1 iu/kg = 2% increase.

! Joint bleed – 25iu/kg (repeat prn)

! Life threat – 50iu/kg (repeat prn)

! Surgery – 50iu/kg x 1

! Prophylaxis – 25iu/kg qod

! Factor IX – 1iu/kg = 1% increase (plasma) 0.75% (recombinant)

! Joint bleed – 50iu/kg (repeat prn)

! Life threat – 100iu/kg (repeat prn)

! Surgery – 100iu/kg x 1

! Prophylaxis – 50iu/kg 2x/wk.

Indication or Site of Bleeding Factor level Desired, % FVIII Dose, IU/kg* Comment

Severe epistaxis; mouth, lip, tongue, or dental work 20-50 10-25 Consider aminocaproic acid (Amicar), 1-2 d

Joint (hip or groin) 40 20 Repeat transfusion in 24-48 h

Soft tissue or muscle 20-40 10-20 No therapy if site small and not enlarging (transfuse if enlarging)

Muscle (calf and forearm) 30-40 15-20 None

Muscle deep (thigh, hip, iliopsoas) 40-60 20-30 Transfuse, repeat at 24 h, then as needed

Neck or throat 50-80 25-40 None

Hematuria 40 20 Transfuse to 40% then rest and hydration

Laceration 40 20 Transfuse until wound healed

GI or retroperitoneal bleeding 60-80 30-40 None

Head trauma (no evidence of CNS bleeding) 50 25 None

Head trauma (probable or definite CNS bleeding, eg, headache, vomiting, neurologic signs) 100 50

Maintain peak and trough factor levels at 100% and 50% for 14 d if CNS bleeding documented†

Trauma with bleeding, surgery† 80-100 50 10-14 d

Acquired Factor VIII antibodies

! Occurs in about 30% of Hemophillia A patients.

! Severity measured in Bethesda units.

! Occur more often with use of recombinant products than plasma based products (45% vs 27% - NEJM 5/26/16).

! Treatment options: 1. Bypassing agents (i.e. Factor VIIa or prothrombin complex concentrates). 2. Induce immune tolerance ( works about 70% of the time). 3. Emicizumab (bispecific antibody which mimics cofactor functionof FVIII)?

! Also holds true for Acquired Hemophillia A (1/1.3 million)

! Acquired antibodies to FVIII in patients without Hemophillia A at birth.

! Risks – increased age, pregnancy, cancer, auto-immune diseases.

Acquired Factor IX antibodies

! Occurs less often vs. Factor VIII.

! Severity measured in Bethesda units.

! Occur more often with use of recombinant products than plasma based products.

! Treatment options: the same much only about ½ as effective. 1. Bypassing agents (i.e. Factor VIIa or prothrombin complex concentrates). 2. Induce immune tolerance 3. Emicizumab (bispecific antibody which mimics cofactor functionof FVIII)?

! Rituximab can also be tried if needed.

Coagulation screening tests in congenital deficienciesPlatlet count

PT APTT PFA TT Congenital Deficiency

N N N N N XIII, mild deficiency of any factor, plasminogen activator inhibitor-1, α2 anti-plasmin

N A N N N VII

N N A N N XII, XI, IX, VIII, prekallikrein, high molecular weight kininogen

N A A N N X, V, II

N A A N A Fibrinogen

N N A or N A or N N Von Willibrands

Factor XIII deficiency

! Factor XIII consists of two non-identical-polypeptide subunits, the "a" chain and the "b“.

! The activated form of factor XIII catalyzes the formation of covalent bonds between the gamma chains and alpha chains of fibrin (cross-linking).

! Cross-linking by Factor XIII does not affect PT and APTT.

! homozygous-recessive patients lack the “a” chains.

! Ecchymoses, hematomas, hemarthroses, and prolonged bleeding after superficial wounds, dental extractions or surgery.

! Tocilizumab Induced Acquired Factor XIII Deficiency. ! Of note: there are 2 factor XIII products on the market.

Acquired coagulopathy

! Numerous acquired conditions and medications

! Liver disease, Alcoholism, or even simple aging. ! Numerous medications can affect coagulopathy

! Acquired conditions – DIC, TTP, Sepsis, ITP, Cancers, etc.

! See a Hematologist and/or correct the underlining cause.

Thank YouQuestions?